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The coronary perivascular adipose tissue (cPVAT) has been associated to the burden of cardiovascular risk factors and to the underlying vessel atherosclerotic plaque severity. Although the "outside to inside" hypothesis of PVAT-derived-adipokine regulation of vessel function is currently accepted, whether the resident mesenchymal stem cells (ASCs) in PVAT have a regulatory role on the underlying vascular arterial smooth muscle cells (VSMCs) is not known. Here, we investigated the interactions between resident PVAT-ASCs and VSMCs. ASCs were obtained from PVAT overlying the left anterior descending (LAD) coronary artery of hearts removed at heart transplant operations. PVAT was obtained both from patients with non-ischemic and ischemic heart disease as the cause of heart transplant. ASCs were isolated from PVAT, phenotypically characterized by flow cytometry, functionally tested for proliferation, and differentiation. Crosstalk between ASCs and VSMCs was investigated by co-culture studies. ASCs were detected in the adventitia of the LAD-PVAT showing differentiation capacity and angiogenic potential. ASCs obtained from PVAT of non-ischemic and ischemic hearts showed different tissue factor (TF) expression levels, different VSMCs recruitment capacity through the axis ERK1/2-ETS1 signaling and different angiogenic potential. Induced upregulation of TF in ASCs isolated from ischemic PVAT rescued their angiogenic capacity in subcutaneously implanted plugs in mice, whereas silencing TF in ASCs decreased the proangiogenic capacity of non-ischemic ASCs. The results indicate for the first time a novel mechanism of regulation of VSMCs by PVAT-ASCs in angiogenesis, mediated by TF expression in ASCs. Regulation of TF in ASCs may become a therapeutic intervention to increase cardiac protection., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

The development of obesity is associated with substantial modulation of adipose tissue (AT) structure. The plasticity of the AT is reflected by its remarkable ability to expand or reduce in size throughout the adult lifespan, which is linked to the development of its vasculature. This increase in AT vasculature could be mediated by the differentiation of adipose tissue-derived stem cells (ASCs) into endothelial cells (ECs) and form new microvasculature. We have already shown that microRNA (miRNA)-145 regulates the differentiation of ASCs into EC-like (ECL) cells. Here, we investigated whether ASCs-differentiation into ECs is governed by a miRNAs signature that depends on fat depot location and /or the metabolic condition produced by obesity. Human ASCs, which were obtained from white AT by surgical procedures from lean and obese patients, were induced to differentiate into ECL cells. We have identified that miRNA-29b-3p in both subcutaneous (s)ASCs and visceral ASCs and miRNA-424-5p and miRNA-378a-3p in subcutaneous (s)ASCs are involved in differentiation into EC-like cells. These miRNAs modulate their pro-angiogenic effects on ASCs by targeting FGFR1 , NRP2 , MAPK1 , and TGF-β2 , and the MAPK signaling pathway. We show for the first time that miRNA-29b-3p upregulation contributes to ASCs' differentiation into ECL cells by directly targeting TGFB2 in both sASCs and visceral ASCs. Moreover, our results reveal that, independent of sASCs' origin (obese/lean), the upregulation of miRNA-378a-3p and the downregulation of miRNA-424-5p inhibit MAPK1 and overexpress FGFR1 and NRP2 , respectively. In summary, both the adipose depot location and obesity affect the differentiation of resident ASCs through the expression of specific miRNAs.

Critical limb ischemia incidence and prevalence have increased over the years. However, there are no successful treatments to improve quality of life and to reduce the risk of cardiovascular and limb events in these patients. Advanced regenerative therapies have focused their interest on the generation of new blood vessels to repair tissue damage through the use of stem cells. One of the most promising sources of stem cells with high potential in cell-based therapy is adipose-derived stem cells (ASCs). ASCs are adult mesenchymal stem cells that are relatively abundant and ubiquitous and are characterized by a multilineage capacity and low immunogenicity. The proangiogenic benefits of ASCs may be ascribed to: (a) paracrine secretion of proangiogenic molecules that may stimulate angiogenesis; (b) secretion of microvesicles/exosomes that are also considered as a novel therapeutic prospect for treating ischemic diseases; and (c) their differentiation capability toward endothelial cells (ECs). Although we know the proangiogenic effects of ASCs, the therapeutic efficacy of ASCs after transplantation in peripheral artery diseases patients is still relatively low. In this review, we evidence the potential therapeutic use of ASCs in ischemic regenerative medicine. We also highlight the main challenges in the differentiation of these cells into functional ECs. However, significant efforts are still needed to ascertain relevant transcription factors, intracellular signaling and interlinking pathways in endothelial differentiation.

Competing Interests: None declared.

Obesity is associated with metabolic disorders such as insulin resistance and type 2 diabetes mellitus (T2DM), further increasing an already heightened cardiovascular risk. Here, amongst obese class III bariatric surgery patients, we have investigated the effect of T2DM in serum and in two, same patient, adipose tissue (AT) depots through proteomic profile expression analyses. Serum and AT samples from subcutaneous (SAT) and visceral (VAT) fat were collected during bariatric surgery. Bead-based targeted multiplex assay systems were used to simultaneously detect and quantify multiple targets in serum samples (targeted proteomics) and analyze changes in adipokine serum composition. AT samples were assessed through an untargeted proteomics approach. Through a systems biology analysis of the proteomic data, information on the affected biological pathways was acquired. In obese class III individuals, the presence of T2DM induced a significantly higher systemic release of ghrelin, GLP-1, glucagon, MMP3, BAFF, chitinase 3-like 1, TNF-R1 and TNF-R2, and a lower systemic release of IL-8. SAT and VAT proteomes belonging to the same patient showed significant differences in local protein content. While the proteins upregulated in VAT were indicative of metabolic dysregulation, SAT protein upregulation suggested adequate endocrine regulation. The presence of T2DM significantly affected VAT protein composition through the upregulation of dysregulating metabolic pathways, but SAT protein composition was not significantly modified. Our results show that T2DM induces metabolic dysregulation in obese individuals with changes in systemic marker levels and impairment of proteostasis in VAT but not in SAT.

Objectives: We performed a comprehensive assessment of the effect of myocardial ischemia duration on cardiac structural and functional parameters by serial cardiac magnetic resonance (CMR) and characterized the evolving scar., Background: CMR follow-up on the cardiac impact of time of ischemia in a closed-chest animal model of myocardial infarction with human resemblance is missing., Methods: Pigs underwent MI induction by occlusion of the left anterior descending (LAD) coronary artery for 30, 60, 90 or 120 min and then revascularized. Serial CMR was performed on day 3 and day 42 post-MI. CMR measurements were also run in a sham-operated group. Cellular and molecular changes were investigated., Results: On day 3, cardiac damage and function were similar in sham and pigs subjected to 30 min of ischemia. Cardiac damage (oedema and necrosis) significantly increased from 60 min onwards. Microvascular obstruction was extensively seen in animals with ≥90 min of ischemia and correlated with cardiac damage. A drop in global systolic function and wall motion of the jeopardized segments was seen in pigs subjected to ≥60 min of ischemia. On day 42, scar size and cardiac dysfunction followed the same pattern in the animals subjected to ≥60 min of ischemia. Adverse left ventricular remodelling (worsening of both LV volumes) was only present in animals subjected to 120 min of ischemia. Cardiac fibrosis, myocyte hypertrophy and vessel rarefaction were similar in the infarcted myocardium of pigs subjected to ≥60 min of ischemia. No changes were observed in the remote myocardium., Conclusion: Sixty-minute LAD coronary occlusion already induces cardiac structural and functional alterations with longer ischemic time (120 min) causing adverse LV remodelling., (© 2022 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of global mortality. Extracellular vesicles (EVs) are small phospholipid vesicles that convey molecular bioactive cargoes and play essential roles in intercellular communication and, hence, a multifaceted role in health and disease. The present review offers a glimpse into the current state and up-to-date concepts on EV field. It also covers their association with several cardiovascular risk factors and ischemic conditions, being subclinical atherosclerosis of utmost relevance for prevention. Interestingly, we show that EVs hold promise as prognostic and diagnostic as well as predictive markers of ASCVD in the precision medicine era. We then report on the role of EVs in atherothrombosis, disentangling the mechanisms involved in the initiation, progression, and complication of atherosclerosis and showing their direct effect in the context of arterial thrombosis. Finally, their potential use for therapeutic intervention is highlighted., (© 2022 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)

Aims: Despite increasing evidence that monocytes may acquire endothelial features, it remains unclear how monocytes participate in angiogenesis after ischaemic damage. We investigated whether ischaemic cells can release microvesicles (MVs) and promote neovascularization in a model of peripheral artery disease (PAD)., Methods and Results: To model PAD, we used an in vivo experimental model of hind-limb ischaemia (HLI) in mice. MVs were isolated from the ischaemic muscle and from peripheral blood at different times after unilateral femoral artery ligation. MVs were phenotypically characterized to identify cell origin. HLI in mice induced the release of MVs with a much higher content of tissue factor (TF) than non-HLI control mice both in the MVs isolated from the affected limb muscle area and from blood. MVs were mainly released from endothelial cells (ECs) and induced Mo differentiation to endothelial cell-like (ECL) cells. Differentiation to ECL cells encompassed highly strict hierarchical transcription factor activation, initiated by ETS1 activation. MVs secreted by microvascular ECs over-expressing TF (upTF-EMVs), were injected in the ischaemic hind-limb in parallel with control EMVs (from random siRNA-treated cells) or EMVs released by silenced TF ECs. In animals treated with upTF-EMVs in the ischaemic zone, there was a highly significant increase in functional new vessels formation (seen by magnetic resonance angiography), a concomitant increase in the pool of circulating Ly6Clow Mo expressing vascular EC markers, and a significantly higher number of Mo/macrophages surrounding and integrating the newly formed collaterals., Conclusion: Ischaemia-activated ECs release EMVs rich in TF that induce monocyte differentiation into ECL cells and the formation of new vessels in the ischaemic zone. TF by this mechanism of formation of new blood microvessels can contribute to ischaemic tissue repair., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

We have recently shown that in ischemic tissue, the hypoxic endothelial cells (EC) release extracellular microvesicles (EMVs) that are rich in tissue factor (TF). These TF-EMVs induce monocyte (Mo) homing to the ischemic zone, their differentiation into EC-like cells, and the formation of new blood vessels increasing tissue perfusion. In addition to membrane proteins, EMVs contain noncoding RNAs that can modulate cellular signaling pathways in the recipient cells. Here, we have investigated whether miRNA contained into secreted EMVs may be transferred into Mo where they could modulate EC-like cell differentiation and angiogenic responses. Our results indicated that EMVs released from activated ECs contain high levels of miR-126 and that the levels are directly proportional to TF expression in EMVs. Interestingly, miR-126 is transferred to Mo when they are incubated with TF-EMVs. Increased levels of miR-126 in Mo do not promote EC-like cell differentiation but regulate angiogenesis by targeting several components of the VEGF pathway, as SPRED1 and PI3KR2. Our findings reveal that activated ECs secrete EMVs carrying miR-126, which can modulate Mo reprogramming of angiogenic genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arderiu, Peña, Civit-Urgell and Badimon.)

Background: The increase in the incidence of obesity and obesity-related cardiovascular risk factors (CVRFs) over the last decades has brought attention on adipose tissue (AT) pathobiology. The expansion of AT is associated with the development of new vasculature needed to perfuse the tissue; however, not all fat depots have the same ability to induce angiogenesis that requires recruitment of their own endothelial cells. In this study we have investigated the effect of different CVRFs, on the angiogenic capacity of the subcutaneous (SAT) and visceral (VAT) adipose tissue and on the function of their mesenchymal cell reservoir., Methods: A transcriptomic approach was used to compare the different angiogenic and inflammatory profiles of the subcutaneous and visceral fat depots from individuals with obesity, as well as their resident stem cells (ASCs). Influence of other risk factors on fat composition was also measured. Finally, the microvesicles (MVs) released by ASCs were isolated and their regenerative potential analyzed by molecular and cellular methodologies., Results: Obesity decreases the angiogenic capacity of AT. There are differences between SAT and VAT; from the 21 angiogenic-related genes analyzed, only three were decreased in SAT compared with those decreased in VAT. ASCs isolated from both fat depots showed significant differences; there was a significant up-regulation of the VEGF-pathway on visceral derived ASCs. ASCs release MVs that stimulate endothelial cell migration and angiogenic capacity., Conclusions: In patients with obesity, SAT expresses a greater number of angiogenic molecules than VAT, independent of the presence of other CVRFs., Competing Interests: The authors declare no conflict of interest.

Aims: High-density lipoproteins (HDLs) are circulating micelles that transport proteins, lipids, and miRNAs. HDL-transported miRNAs (HDL-miRNAs) have lately received attention but their effects on vascular cells are not fully understood. Additionally, whether cardiovascular risk factors affect HDL-miRNAs levels and miRNA transfer to recipient cells remains equally poorly known. Here, we have investigated the changes induced by hypercholesterolaemia on HDL-miRNA levels and its effect on recipient endothelial cells (ECs)., Methods and Results: Pigs were kept on a high-fat diet (HC; n = 10) or a normocholesterolaemic chow (NC; n = 10) for 10 days reaching cholesterol levels of 321.0 (229.7-378.5) mg/dL and 74.0 (62.5-80.2) mg/dL, respectively. HDL particles were isolated, purified, and quantified. HDL-miRNA profiling (n = 149 miRNAs) of HC- and NC-HDLs was performed by multipanel qPCR. Cell cultures of porcine aortic ECs were used to determine whether HDL-miRNAs were delivered to ECs. Potential target genes modulated by miRNAs were identified by bioinformatics and candidate miRNAs were validated by molecular analysis. In vivo effects in the coronary arteries of normocholesterolaemic swine administered HC- or NC-HDLs were analysed. Among the HDL-miRNAs, four were found in different amounts in HC- and NC-HDL (P < 0.05). miR-126-5p and -3p and miR-30b-5p (2.7×, 1.7×, and 1.3×, respectively) were found in higher levels and miR-103a-3p and miR-let-7g-5p (-1.6×, -1.4×, respectively) in lower levels in HC-HDL. miR-126-5p and -3p were transferred from HC-HDL to EC (2.5×; P < 0.05), but not from NC-HDL, by a SRB1-mediated mechanism. Bioinformatics revealed that HIF1α was the miR-126 target gene with the highest predictive value, which was accordingly found to be markedly reduced in HC-HDL-treated ECs and in miR126 mimic transfected ECs. In vivo validation confirmed that HIF1α was diminished in the coronary endothelial layer of NC pigs administered HC-HDL vs. those administered NC-HDL (P < 0.05)., Conclusion: Hypercholesterolaemia induces changes in the miRNA content of HDL enhancing miR126 and its delivery to ECs with the consequent down-regulation of its target gene HIF1α., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: The composition and function of the adipose tissue covering the heart are poorly known. In this study, we have investigated the epicardial adipose tissue (EAT) covering the cardiac ventricular muscle and the EAT covering the left anterior descending artery (LAD) on the human heart, to identify their resident stem cell functional activity., Methods: EAT covering the cardiac ventricular muscle was isolated from the apex (avoiding areas irrigated by major vessels) of the heart (ventricular myocardium adipose tissue (VMAT)) and from the area covering the epicardial arterial sulcus of the LAD (PVAT) in human hearts excised during heart transplant surgery. Adipose stem cells (ASCs) from both adipose tissue depots were immediately isolated and phenotypically characterized by flow cytometry. The different behavior of these ASCs and their released secretome microvesicles (MVs) were investigated by molecular and cellular analysis., Results: ASCs from both VMAT (mASCs) and the PVAT (pASCs) were characterized by the expression of CD105, CD44, CD29, CD90, and CD73. The angiogenic-related genes VEGFA, COL18A1, and TF, as well as the miRNA126-3p and miRNA145-5p, were analyzed in both ASC types. Both ASCs were functionally able to form tube-like structures in three-dimensional basement membrane substrates. Interestingly, pASCs showed a higher level of expression of VEGFA and reduced level of COL18A1 than mASCs. Furthermore, MVs released by mASCs significantly induced human microvascular endothelial cell migration., Conclusion: Our study indicates for the first time that the resident ASCs in human epicardial adipose tissue display a depot-specific angiogenic function. Additionally, we have demonstrated that resident stem cells are able to regulate microvascular endothelial cell function by the release of MVs.

Rationale: Adipose-derived stem cells (ASCs) are a potential adult mesenchymal stem cell source for restoring endothelial function in ischemic tissues. However, the mechanism that promotes ASCs differentiation toward endothelial cells (ECs) is not known., Objective: To investigate the mechanisms of ASCs differentiation into ECs., Methods and Results: ASCs were isolated from clinical lipoaspirates and cultured with DMEM or endothelial cell-conditioned medium. Endothelial cell-conditioned medium induced downregulation of miR-145 in ASCs and promoted endothelial differentiation. We identified bFGF (basic fibroblast growth factor) released by ECs as inducer of ASCs differentiation through receptor-induced AKT (protein kinase B) signaling and phosphorylation of FOXO1 (forkhead box protein O1) suppressing its transcriptional activity and decreasing miR-145 expression. Blocking bFGF-receptor or PI3K/AKT signaling in ASCs increased miR-145 levels. Modulation of miR-145 in ASCs, using a miR-145 inhibitor, regulated their differentiation into ECs: increasing proliferation, migration, inducing expression of EC markers (VE-cadherin, VEGFR2 [vascular endothelial growth factor receptor 2], or VWF [von Willebrand Factor]), and tube-like formation. Furthermore, in vivo, downregulation of miR-145 in ASCs enhanced angiogenesis in subcutaneously implanted plugs in mice. In a murine hindlimb ischemia model injection of ASCs with downregulated miR-145 induced collateral flow and capillary formation evidenced by magnetic resonance angiography. Next, we identified ETS1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1) as the target of miR-145. Upregulation of miR-145 in ASCs, by mimic miR-145, suppressed ETS1 expression and consequently abolished EC differentiation and the angiogenic properties of endothelial cell-conditioned medium-preconditioned ASCs; whereas, overexpression of ETS1 reversed the abrogated antiangiogenic capacity of miR-145. ETS1 overexpression induced similar results to those obtained with miR-145 knockdown., Conclusions: bFGF released by ECs induces ASCs differentiation toward ECs through miR-145-regulated expression of ETS1. Downregulation of miR-145 in ASCs induce vascular network formation in ischemic muscle.

C-reactive protein (CRP) is a short pentraxin mainly found as a pentamer in the circulation, or as non-soluble monomers CRP (mCRP) in tissues, exerting different functions. This review is focused on discussing the role of CRP in cardiovascular disease, including recent advances on the implication of CRP and its forms specifically on the pathogenesis of atherothrombosis and angiogenesis. Besides its role in the humoral innate immune response, CRP contributes to cardiovascular disease progression by recognizing and binding multiple intrinsic ligands. mCRP is not present in the healthy vessel wall but it becomes detectable in the early stages of atherogenesis and accumulates during the progression of atherosclerosis. CRP inhibits endothelial nitric oxide production and contributes to plaque instability by increasing endothelial cell adhesion molecules expression, by promoting monocyte recruitment into the atheromatous plaque and by enzymatically binding to modified low-density lipoprotein. CRP also contributes to thrombosis, but depending on its form it elicits different actions. Pentameric CRP has no involvement in thrombogenesis, whereas mCRP induces platelet activation and thrombus growth. In addition, mCRP has apparently contradictory pro-angiogenic and anti-angiogenic effects determining tissue remodeling in the atherosclerotic plaque and in infarcted tissues. Overall, CRP contributes to cardiovascular disease by several mechanisms that deserve an in-depth analysis.

Atherosclerosis (AT) is a progressive chronic disease involving lipid accumulation, fibrosis, and inflammation in medium and large-sized arteries, and it is the main cause of cardiovascular disease (CVD). AT is caused by dyslipidemia and mediated by both innate and adaptive immune responses. Despite lipid-lowering drugs have shown to decrease the risk of cardiovascular events (CVEs), there is a significant burden of AT-related morbidity and mortality. Identification of subjects at increased risk for CVE as well as discovery of novel therapeutic targets for improved treatment strategies are still unmet clinical needs in CVD. Microvesicles (MVs), small extracellular plasma membrane particles shed by activated and apoptotic cells have been widely linked to the development of CVD. MVs from vascular and resident cells by facilitating exchange of biological information between neighboring cells serve as cellular effectors in the bloodstream and play a key role in all stages of disease progression. This article reviews the current knowledge on the role of MVs in AT and CVD. Attention is focused on novel aspects of MV-mediated regulatory mechanisms from endothelial dysfunction, vascular wall inflammation, oxidative stress, and apoptosis to coagulation and thrombosis in the progression and development of atherothrombosis. MV contribution to vascular remodeling is also discussed, with a particular emphasis on the effect of MVs on the crosstalk between endothelial cells and smooth muscle cells, and their role regulating the active process of AT-driven angiogenesis and neovascularization. This review also highlights the latest findings and main challenges on the potential prognostic, diagnostic, and therapeutic value of cell-derived MVs in CVD. In summary, MVs have emerged as new regulators of biological functions in atherothrombosis and might be instrumental in cardiovascular precision medicine; however, significant efforts are still needed to translate into clinics the latest findings on MV regulation and function.

Essentials Monocytes (Mo) transdifferentiate into endothelial cell-like (ECL) cells. Mo induce tissue factor (TF) expression and secretion in microvascular endothelial cells (mECs). TF interacts with Mo in a paracrine fashion, inducing their transdifferentiation into ECL cells. TF generates a positive feedback crosstalk between Mo and mECs that promotes angiogenesis., Summary: Background Monocytes (Mo) increase neovascularization by releasing proangiogenic mediators and/or transdifferentiating into endothelial cell-like (ECL) cells. Recently, we have reported that Mo-microvascular endothelial cells (mECs) crosstalk induces mEC-tissue factor (TF) expression and promotes angiogenesis. However, the effect of TF on Mo remains unknown. Objective Here, we analyzed whether TF might exert angiogenic effects by inducing transdifferentiation of Mo. Methods Full-length TF (flTF) and alternatively spliced TF (asTF) were overexpressed in mECs, and their supernatants were added to Mo cultures. CD16 positivity and expression of vascular endothelial cell (VEC) markers in Mo were analyzed by fluorescence activated cell sorting. The capacity to form tube-like structures were visualized in three-dimensional cultures. Results In mECs flTF and asTF expression and release were increased in cultures with Mo-conditioned media. TF variants induced expansion of a CD16 + Mo subset and Mo transdifferentiation into ECL-cells expressing VEC markers that can form new microvessels. CD16 + Mo exposed to TF showed an increased expression of VE-cadherin, von Willebrand factor (VWF) and eNOS. Mo cultured with supernatants obtained from TF-silenced mECs did not transdifferentiate to ECL-cells or expressed VEC markers. Blocking β1-integrin in Mo significantly blocked the effects of the TF variants. Conclusions Mo induce mECs to express and release TF, which drives CD16 - Mo to transform into CD16 + Mo and to transdifferentiate into ECL-cells that can form new microvessels. Our results reveal a TF-mediated positive feedback between mECs and Mo that stimulates Mo differentiation and induces angiogenesis., (© 2017 International Society on Thrombosis and Haemostasis.)

Background: Myocardial microvascular loss after myocardial infarction (MI) remains a therapeutic challenge. Autologous stem cell therapy was considered as an alternative; however, it has shown modest benefits due to the impairing effects of cardiovascular risk factors on stem cells. Allogenic adipose-derived stem cells (ASCs) may overcome such limitations, and because of their low immunogenicity and paracrine potential may be good candidates for cell therapy. In the present study we investigated the effects of allogenic ASCs and their released products on cardiac rarefaction post MI., Methods: Pig subcutaneous adipose tissue ASCs were isolated, expanded and GFP-labeled. ASC angiogenic function was assessed by the in-vivo chick chorioallantoic membrane (CAM) model. Pigs underwent MI induction and 7 days after were randomized to receive: allogenic ASCs (intracoronary infusion); conditioned media (CM; intravenous infusion); ASCs + CM; or PBS/placebo (control). Cardiac damage and function were monitored by 3-T cardiac magnetic resonance imaging upon infusion (baseline CMR) and 1 and 3 weeks thereafter. We assessed in the myocardium: microvessel density; angiogenic markers (CD105, CD31, TF, VEGFR2, VEGFR1, vWF, eNOS, CD62); collagen deposition; and reparative fibrosis (TGFβ/TβRII/collagen). Differential proteomics of ASCs and CM was performed to characterize the ASC protein signature., Results: CAM indicated a significant ASC proangiogenic capacity. In pigs after MI, only PBS/placebo animals displayed an impaired cardiac function 3 weeks after infusion (p < 0.05 vs baseline). Administration of ASCs + CM significantly enhanced neovessel formation and favored cardiac repair post MI (p < 0.05 vs the other groups). Molecular markers of angiogenesis were significantly upregulated both at transcriptional and protein levels (p < 0.05). The in-silico bioinformatics analysis of the ASC and CM proteome (interactome) indicated activation of a coordinated protein network involved in the formation of microvessels and the resolution of rarefaction., Conclusion: Coadministration of allogenic ASCs and their CM synergistically contribute to the neovascularization of the infarcted myocardium through a coordinated upregulation of the proangiogenic protein interactome.

Inflammation contributes to vascular disease progression. However, the role of circulating inflammatory molecules on microvascular endothelial cell (mECs) is not fully elucidated. The aim of this study was to investigate the effects of the short pentraxin CRP on microvascular endothelial cell angiogenic function. Subcutaneously implanted collagen plugs seeded with human mECs exposed to monomeric CRP (mCRP) in mice showed formation of an extended network of microvessels both in the plug and the overlying skin tissue, while mECs exposure to pentameric native CRP (nCRP) induced a much milder effect. To understand the mechanisms behind this angiogenic effects, mECs were exposed to both CRP forms and cell migration, wound repair and tube-like formation were investigated. nCRP effects were moderate and of slow-onset whereas mCRP induced rapid, and highly significant effects. We investigated how circulating nCRP is transformed into mCRP by confocal microscopy and western blot. nCRP is transformed into mCRP on the mECs membranes in a time dependent fashion. This transformation is specific and in part receptor dependent, and the formed mCRP triggers F3 gene transcription and TF-protein expression in mECs to induce angiogenesis. F3-silenced mECs are unable to form angiotubes. In agreement, mCRP induced upregulation of the TF signalling pathway in mECs with downstream phosphorylation of AKT and activation of the transcription factor ETS1 leading to increased CCL2 release. The circulating pentraxin nCRP with little pro-angiogenic effect when dissociated into mCRP on the surface of mECs is able to trigger potent proangiogenic effects by inducing F3-gene upregulation and TF signalling.

Circulating microparticles (cMPs) are small phospholipid-rich microvesicles shed by activated cells that play a pivotal role in cell signalling related to the pathogenesis of atherothrombosis. We aimed to investigate the prognostic value of cMPs released from different vascular cells for cardiovascular event (CVE) presentation in asymptomatic patients at high cardiovascular risk factors under nutritional and pharmacologic treatment. This is a nested case-control study of 50 patients from the five-year follow-up prospective PREDIMED trial enrolled in the nuts arm of the Mediterranean diet (MedDiet-nuts). We randomly selected 25 patients who had suffered a CVE during follow-up and pair-matched them for sex, age, and classical CV risk factors to 25 patients who remained asymptomatic (no-CVE). Total Annexin V-(AV)+ cMPs and cMPs from cells of the vascular compartment were quantified by flow cytometry at baseline and after one year follow-up. MedDiet-nuts and pharmacological treatment neither modified levels nor source of MP shedding in CVE patients. However, no-CVE patients showed 40-86 % decreased total AV+, PAC-1+/AV+, CD61+/AV+, CD142+/CD61+/AV+, CD62P+/AV+, CD146+/AV+, CD63+/AV+ and CD11a+/AV+ cMPs at one year follow-up (p≤0.046, all). CD142+/CD61+/AV+, CD146+/AV+ and CD45+/AV+ cMPs were decreased in no-CVE patients compared to CVE patients. A ROC-curve clustered model for CD142+/CD61+/AV+, CD45+/AV+ and CD146+/AV+ cMPs predicted a future CVE [p<0.0001, AUC=0.805 (0.672 to 0.938)]. In patients at high CV risk profile treated with a controlled MedDiet supplemented with nuts and receiving up-to-date CV drug treatment, reduced cMPs derived from activated platelets, leukocytes and endothelial cells are predictive of protection against CVE within the next four years.

Tissue factor (TF) signaling regulates gene expression and protein synthesis leading to the modulation of cell function. Recently, we have demonstrated in microvascular endothelial cells (mECs) that TF signaling induces activation of ETS1 transcription factor. Because combinatorial control is a characteristic property of ETS family members, involving the interaction between ETS1 and other transcription factors, here we investigate whether additional transcription factors are involved in TF-induced angiogenesis. We show by in vitro and in vivo experiments that in addition to ETS1, SMAD3 contributes to tube-like stabilization induced by TF in mECs. Whereas the ability of TF-overexpressing cells to induce gene expression through ETS1 is dependent on AKT signaling, SMAD3 induces ETS1 by an alternative AKT-independent pathway. Moreover, while TF-AKT-ETS1 pathway to induce CCL2 is PAR2-independent, PAR2 is required for TF-SMAD3-induced CCL2 expression. PAR2-dependent activation of SMAD3 is mediated by PKC phosphorylation. In addition, disruption of SMAD3 expression in mECs reduces ERK1/2 phosphorylation and decreases target gene promoter activity. In conclusion, in mECs TF-induced angiogenesis seems to be the result of two signaling pathways: TF-induced microvessel formation is regulated through β1 integrin-AKT-ETS1; and TF-induced microvessel stabilization is regulated via PAR2-SMAD3 that is indispensable for the maintenance of vascular integrity., (© The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

Protein-disulphide isomerase family (PDI) are an ER-stress protein that controls TF-procoagulant activity but its role in HVSMC migration and coronary artery disease remains to be elucidated. We aimed to investigate whether in human coronary smooth muscle cells (HVSMC) the ER-stress protein-disulphide isomerase family A member 2 (PDIA2) regulates tissue factor (TF) polarisation during migration and atherosclerotic remodeling. PDIA2 and TF were analysed by confocal microscopy, silenced by small interfering RNAs (siRNA) and their function analysed by transwell and migration assays in vitro and in vivo. PDIA2and TF co-localise in the front edge of motile HVSMC. Silencing PDIA2, as well as silencing TF, reduces migration. PDIA2 silenced cells show increased TF-rich microparticle shedding. In vivo cell-loaded plug implants in nude mice of PDIA2 silenced HVSMC together with microvascular endothelial cells showed a significant impairment in mature microvessel formation. PDIA2 and TF are found in remodelled atherosclerotic plaques but not in healthy coronaries. In conclusion, we demonstrate that TF is chaperoned by PDIA2 to the HVSMC membrane and to the cell migratory front. Absence of PDIA2 impairs TF intracellular trafficking to its membrane docking favoring its uncontrolled release in microparticles. TF-regulated HVSMC migration and microvessel formation is under the control of the ER-protein PDIA2.

Objective: Therapeutic angiogenesis is a promising strategy for treating ischemia. Our previous work showed that endogenous endothelial tissue factor (TF) expression induces intracrine signaling and switches-on angiogenesis in microvascular endothelial cells (mECs). We have hypothesized that activated mECs could exert a further paracrine regulation through the release of TF-rich microvascular endothelial microparticles (mEMPs) and induce neovascularization of ischemic tissues., Approach and Results: Here, we describe for the first time that activated mECs are able to induce reparative neovascularization in ischemic zones by releasing TF-rich microparticles. We show in vitro and in vivo that mEMPs released by both wild-type and TF-upregulated-mECs induce angiogenesis and collateral vessel formation, whereas TF-poor mEMPs derived from TF-silenced mECs are not able to trigger angiogenesis. Isolated TF-bearing mEMPs delivered to nonperfused adductor muscles in a murine hindlimb ischemia model enhance collateral flow and capillary formation evidenced by MRI. TF-bearing mEMPs increase angiogenesis operating via paracrine regulation of neighboring endothelial cells, signaling through the β1-integrin pathway Rac1-ERK1/2-ETS1 and triggering CCL2 (chemokine [C-C motif] ligand 2) production to form new and competent mature neovessels., Conclusions: These findings demonstrate that TF-rich mEMPs released by microvascular endothelial cells can overcome the consequences of arterial occlusion and tissue ischemia by promoting postischemic neovascularization and tissue reperfusion., (© 2014 American Heart Association, Inc.)

Angiogenesis during reactive and pathologic processes is characteristically associated with inflammation. Inflammatory cells participate in angiogenesis by secreting different molecules that affect endothelial cell functions. We had previously shown that induced tissue factor (TF) expression in activated microvascular endothelial cells (mEC) is able to induce angiogenesis via autocrine regulation. However, the signals that induce TF expression in mEC are not fully known. Here, we demonstrate that monocyte paracrine cross-talk with mECs triggers mEC-TF expression. We have identified that monocyte-secreted Wnt5a induces TF expression in mEC and functionally induces cell monolayer repair and angiotube formation in vitro as well as microvessel formation in vivo. Monocyte-secreted Wnt5a activates FZD5 in mECs, which signals to induce the release of intracellular Ca(2+) and increase NFκB transcription activity and TF gene expression. In sum, Wnt5a secreted by monocytes signals through the noncanonical Wnt-FZD5 pathway in mECs to induce TF expression that induces angiogenesis by autocrine regulation., (© The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

Background: Tissue factor (TF) is the most relevant physiological trigger of thrombosis contributing to the presentation of clinical ischemic events after plaque rupture. However, the role of human vascular smooth muscle cell (HVSMC) TF in vascular remodeling, restenosis and atherosclerosis is less known. We have hypothesized that TF contributes to atherosclerotic lesion formation, triggering smooth muscle cell migration through a specific yet unknown signaling pathway., Objectives: The aim of this study has been to investigate the signal transduction mechanism by which TF may contribute to the transition of resident static contractile HVSMC into a migrating cell that promotes atherosclerotic plaque progression., Methods: We have used a system biology discovery approach with gene-engineered HVSMCs to identify genes/proteins involved in the TF-triggered effects in HVSMC obtained from the coronary arteries of human adult hearts., Results: Analysis of wild-type HVSMC (TF(+) ) and TF(-) silenced HVSMC (TF(-) ) showed that TF is involved in the regulation of Wnt signaling and in the expression of downstream proteins that affect the atherosclerotic process., Conclusions: The 'in silico' analysis pointed to specific Wnt-pathway proteins that have been validated in cell culture and also have been found expressed in human advanced atherosclerotic plaques but not in early lesions. TF signals through Wnt to regulate coronary smooth muscle cell migration and vascular remodeling., (© 2013 International Society on Thrombosis and Haemostasis.)

Tissue factor (TF) has well-recognized roles as initiator of blood coagulation as well as an intracellular signaling receptor. TF signaling regulates gene transcription and protein translation. Recently, we have shown that TF-induced mature neovessel formation is ultimately driven by CCL2 expression. However, the signaling process induced by TF to promote microvessel formation remains to be determined. This study was designed with the objective to investigate the mechanisms involved in TF-induced neovessel formation. Here, we have identified that Ets-1 expression is a downstream effector of TF signaling. TF-siRNA induced a highly significant reduction in Ets-1 expression levels and in Ets-1/DNA binding while inducing abrogation of microvessel formation. Activation of Ets-1 rescued the effect of TF inhibition and restored microvessel formation confirming the critical role of Ets-1 in TF-induced angiogenesis. VE-cadherin expression, a key regulator of endothelial intercellular junctions, and an Ets-1 target molecule was dependent of TF-inhibition. We show that TF signals through ERK1/2 to activate Ets-1 and induce CCL2 gene expression by binding to its promoter region. We conclude that endothelial cell TF signals through ERK1/2 and Ets-1 to trigger microvessel formation.

Background: Tissue factor (TF) is the most relevant physiological trigger of thrombosis. Additionally TF is a transmembrane receptor with cell signaling functions., Objectives: The aim of this study was to investigate TF subcellular localization, function and signaling in human coronary artery smooth muscle cell migration., Methods: Coronary arteries and primary cultures of vascular smooth muscle cells (HVSMC) were obtained from human explanted hearts. Wound repair and Boyden chamber assays were used to measure migration in vitro. TF-pro-coagulant activity (TF-PCA) was measured in extracted cellular membranes. Analysis of TF distribution was performed by confocal microscopy. A nucleofector device was used for TF and protease activated receptor 2 (PAR2) silencing. mRNA levels were analyzed by RT-PCR., Results: In migrating HVSMC TF translocates to the leading edge of the cells showing an intense patch-like staining in the lamellipodia. In the migrating front TF colocalizes with filamin (FLN) in the polarized lipid rafts. TF-PCA was increased in migrating cells. Silencing of the TF gene inhibits RSK-induced FLN-Ser-2152 phosphorylation, down-regulates CDC42, RhoA, and Rac1 protein expression and significantly inhibits cell migration. Silencing PAR2 also inhibits cell migration; however, silencing both TF and PAR2 induces a significantly higher effect on migration. Smooth muscle cells expressing TF have been identified in non-lipid-rich human coronary artery atherosclerotic plaques., Conclusions: TF translocates to the cell front in association with cytoskeleton proteins and regulates HVSMC migration by mechanisms dependent and independent of factor (F)VIIa/PAR2. These results extend the functional role of TF to smooth muscle cell trafficking in vessel wall remodeling., (© 2012 International Society on Thrombosis and Haemostasis.)

Background: Tissue factor (TF) and its signaling mediators play a crucial role in angiogenesis. We have previously shown that TF-induced endothelial cell (EC) CCL2 release contributes to neovessel formation., Objective: In this study, we have investigated the signaling pathways involved in TF-induced EC tube formation., Methods: The human microvascular endothelial cell line (HMEC-1) cultured onto basement membrane-like gel (Matrigel) was used to study TF signaling pathways during neovessels formation., Results: Inhibition of endogenous TF expression in ECs using siRNA resulted in inhibition of a stable tube-like structure formation in three-dimensional cultures, associated with a down-regulation of Akt activation, an increased phosphorylation of Raf at Ser(259) with a subsequent reduction of Raf kinase and a reduction of ERK1/2 phosphorylation ending up in Ets-1 transcription factor inhibition. Conversely, overexpression of TF resulted in an increase in tube formation and up-regulation of Akt protein. Moreover, immunoprecipitation of Akt and western blotting of the immunoprecipitates with anti-TF antibody revealed a direct interaction between TF and Akt. The effects of silencing TF were partially reversed by a PAR2 agonist that rescued tube formation, indicating that the TF-Akt pathway induces PAR2-independent effector signaling. Finally, enforced expression of Akt in TF-silenced ECs rescued tube formation in a Matrigel assay and induced Ets-1 phosphorylation., Conclusions: In EC, TF forms a complex with Akt activating Raf/ERK and Ets-1 signaling induces microvessel formation., (© 2012 International Society on Thrombosis and Haemostasis.)

Objective: Tissue factor (TF) triggers arterial thrombosis. TF is also able to initiate cellular signaling mechanisms leading to angiogenesis. Because high cardiovascular risk atherosclerotic plaques show significant angiogenesis, our objective was to investigate whether TF is able to trigger and stabilize atherosclerotic plaque neovessel formation., Methods and Results: In this study, we showed, by real-time confocal microscopy in 3-dimensional basement membrane cocultures, that TF in human microvascular endothelial cells (HMEC-1) and in human vascular smooth muscle cells (HVSMCs) plays an important role in the formation of capillary-like networks. TF silencing in endothelial cells and smooth muscle cells inhibits the formation of tube-like structures with stable phenotype. Using an in vivo model, we observed that TF inhibition in either HMEC-1 or HVSMCs reduced their shared ability to form new capillaries. The phenotypic changes induced by TF silencing were linked to reduced chemokine (C-C motif) ligand 2 (CCL2) expression in endothelial cells. Wound healing and chemotactic assays demonstrated that TF-induced release of CCL2 stimulated HVSMC migration to HMEC-1., Conclusion: Endogenous TF regulates CCL2 production in endothelial cells. Secreted CCL2 mediates the angiogenic effect of TF by recruiting smooth muscle cells toward endothelial cells and facilitates the maturation of newly formed microvessels.

Platelet activation leads to the initiation of intracellular signalling processes, many of which are triggered by Ca2+. We have studied the involvement of exogenous Ca2+ in platelet response to collagen activation. Platelet suspensions were prepared with and without adding external calcium in the suspension buffers. Activation with collagen (Col-I) was carried out, before and after incubation with cytochalasin B (Cyt-B) to block the actin assembly and the cytoskeletal reorganization. We evaluated changes in (i) tyrosine phosphorylation of proteins, in platelet lysates and associated with the cytoskeletal fraction, (ii) the association of contractile proteins to the cytoskeleton, (iii) expression of intraplatelet substances at the surface, and (iv) cytosolic Ca2+ levels ([Ca2+]i). Ultrastructural evaluation of platelets by electron microscopy was also performed. Platelet activation by Col-I in the absence of added Ca2+ was followed by mild association of actin and other contractile proteins, low phosphorylation of proteins at tyrosine residues, lack of expression of intraplatelet substances at the membrane, and absence of aggregation. In the presence of millimolar Ca2+, Col-I induced intense actin filament formation with association of contractile proteins with the cytoskeleton, resulting in profound morphological changes. Under these conditions, Col-I induced signalling through tyrosine phosphorylation, with increases in the [Ca2+]i, release of intragranule content and aggregation. Inhibiting actin polymerization with Cyt-B prevented all these events. Our data indicates that platelet activation by collagen requires external Ca2+. Studies with Cyt-B indicate that assembly of new actin and cytoskeleton-mediated contraction, both dependent on exogenous Ca2+, are key events for platelet activation by collagen. In addition, our results confirm that entrance of exogenous Ca2+ depends on a functional cytoskeleton.

Background: Deficient hemostasis and accelerated atherosclerosis coexist in patients with chronic kidney disease. Endothelial dysfunction may be involved in the high incidence of atherothrombotic events in these patients. We established an in vitro model of endothelial dysfunction by exposing endothelial cells to uremic media and applied a proteomic approach to characterize endothelial cell dysfunction in uremia., Study Design: Cross-sectional study., Setting and Participants: Serum samples from 8 patients with chronic kidney disease on hemodialysis treatment were collected., Predictor: Exposure of cultured endothelial cells to normal and uremic serum. OUTCOME AND MEASUREMENTS: Proteins from lysed cells were characterized by isoelectric point and molecular weight by using 2-dimensional gel electrophoresis. Spots were visualized by means of silver staining and identified by using mass spectrometry., Results: Identification of the most prominent proteins showed molecules related to inflammation (high mobility group box 1, aldose reductase, and proteasome components) and oxidative stress (superoxide dismutase and glutathione peroxidase), both associated with chronic kidney disease. These changes may be caused by activation of the nuclear factor-kappaB transcription factor. Changes in expression of cytoskeletal proteins (destrin and vimentin) also were detected., Limitations: In vitro study., Conclusion: Proteomic techniques proved to be a powerful tool to investigate endothelial dysfunction in uremia. A more exhaustive analysis will provide answers and potential therapeutic targets in the near future.

Normal vascular development and angiogenesis is regulated by coordinated changes in cell-cell and cell-extracellular matrix (ECM) interactions. The Homeobox (Hox) family of transcription factors coordinately regulate expression of matrix degrading proteinases, integrins and ECM components and profoundly impact vascular remodeling. Whereas HoxA5 is down regulated in active angiogenic endothelial cells (EC), sustained expression of HoxA5 induces TSP-2 and blocks angiogenesis. Since HoxA5 is also lacking in EC in proliferating hemangiomas, we investigated whether restoring expression of HoxA5 could normalize hemangioma cell morphology and/or behavior. Sustained expression of HoxA5 in the murine hemangioma cell line (EOMA) reduced their growth in vivo and promoted branching morphogenesis in 3D BM cultures. Moreover, restoring HoxA5 expression increased the retention of beta-catenin in adherens junctions and reduced permeability. In addition we also show that the HoxA5 mediated increase in stability of adherens junctions requires Akt1 activity and introduction of constitutively active myr-Akt in EOMA cells also increased retention of beta-catenin in adherens junctions. Finally we show that HoxA5 increases Akt1 mRNA, protein expression and further enhances Akt activity via a coordinate down regulation of PTEN. Together these results demonstrate a central role for HoxA5 in coordinating a stable vascular phenotype.

Objective: Licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) has been demonstrated to inhibit cyclooxygenase (COX)-1, COX-2, and 5-lipoxygenase. The aim of this study was to investigate the in-vitro effects of licofelone on platelet function. Effects observed were compared with those produced by the classic COX-1 inhibitor aspirin (ASA)., Methods: Platelet aggregation was assessed by a turbidimetric method. Platelet haemostatic performance was studied with the platelet function analyser (PFA-100), using collagen epinephrine and collagen ADP cartridges. Interaction of platelets with thrombogenic surfaces was analysed by perfusion experiments performed under flow conditions using both parallel and annular chambers., Results: Licofelone prolonged the lag time of platelet aggregation induced by arachidonic acid and reduced maximal platelet aggregation induced by ADP or collagen. Studies using PFA-100 demonstrated that licofelone (0.1, 1 and 10 muM) significantly prolonged closure times (P < 0.05) with both types of cartridges. In studies with the parallel chamber exposing purified collagen, both licofelone and ASA significantly reduced (P < 0.05) overall platelet interaction with the thrombogenic surface. In studies performed in annular chamber exposing a highly thrombogenic vessel surface, licofelone reduced height and area of the platelet masses deposited (7.0 +/- 0.5 mum; P < 0.005 and 80.2 +/- 17.3 mum; P < 0.05 vs. control 10.6 +/- 0.9 mum and 194.8 +/- 44.7 mum, respectively). ASA also impaired thrombus formation but differences did not reach the levels of statistical significance., Conclusions: Under our experimental in-vitro conditions, licofelone interfered with platelet function as demonstrated by a diminished platelet aggregation, being more powerful than ASA and reducing the interaction of platelets with thrombogenic surfaces.

Homeobox (Hox) genes are master regulatory genes that direct organogenesis and maintain differentiated tissue function. We previously reported that HoxD10 helps to maintain a quiescent, differentiated phenotype in endothelial cells by suppressing expression of genes involved in remodeling the extracellular matrix and cell migration. Here we investigated whether HoxD10 could also promote or maintain a differentiated phenotype in epithelial cells. We observed that HoxD10 expression is progressively reduced in epithelial cells as malignancy increases in both breast and endometrial tumors. Retroviral gene transfer to restore expression of HoxD10 in the malignant breast tumor cells MDA-MB-231 significantly impaired migration, and when these cells were cultured in a three-dimensional laminin-rich basement membrane (3DlrBM) model, they formed polarized, acinar structures. This phenotypic reversion was accompanied by decreased alpha3 integrin expression and reduced proliferation. Importantly, expression of HoxD10 in the MDA-MB-231 cells inhibited their ability to form tumors in mouse xenografts. Taken together, our results suggest that HoxD10 has tumor-suppressive functions for mammary epithelial cells.

Background: The ability of nitrous compounds to donate nitric oxide (NO), an agent with vasodilating and inhibitory effects on platelet function, has been considered a useful pharmacologic strategy for cardiovascular complications. The purpose of this study was to investigate the effects of a new NO donor, LA419, on platelet interaction in an ex vivo model with human blood circulating through collagen-rich surfaces., Materials and Methods: Platelet adhesive and cohesive function were analyzed by morphometric procedures after perfusion techniques. Treated blood was exposed to thrombogenic surfaces and platelet interactions were morphometrically evaluated., Results: All the concentrations studied of LA419 (10 microM, 20 microM and 100 microM) reduced overall platelet interaction with a collagen surface (27.19 +/- 4.72; 25.52 +/- 3.52; and 23.44 +/- 3.01, P < 0.05, respectively, vs. 32.31 +/- 1.61% in the control). The antithrombotic effect was confirmed by results in cross-sectional studies performed in arterial vessels exposed to circulating blood. Values of thrombus and covered surface at 20 microM LA419 were, respectively, 13.67 +/- 4.97% and 19.01 +/- 5.89%; respect to controls 34.80 +/- 5.29% and 37.93 +/- 5.34% (P < 0.05). Moreover, LA419 reduced significantly thrombus area (88.45 +/- 21.97 microm(2); P < 0.05) with respect to controls (168.45 +/- 21.97 microm(2)) and thrombus height, from an average of 10.27 +/- 1.05 microm in nontreated blood to 7.16 +/- 0.6 microm in treated samples (P < 0.05)., Conclusion: From the present data we can conclude that LA419 possesses a strong antiplatelet action, as demonstrated by its ability to significantly inhibit the interaction of platelet with highly thrombogenic collagen surfaces.

Background: Homeobox (Hox) genes are transcriptional regulators which modulate embryonic morphogenesis and pathological tissue remodeling in adults via regulation of genes associated with cell-cell or cell extracellular matrix (ECM) interactions. We previously showed that while Hox 3 genes promote angiogenesis, Hox D10 inhibits this process., Methods and Results: Here we show that another Hox family gene, Hox A5, also blocks angiogenesis but accomplishes this by targeting different downstream genes than Hox D10. Sustained expression of Hox A5 leads to down regulation of many pro-angiogenic genes including VEGFR2, ephrin A1, Hif1alpha and COX-2. In addition, Hox A5 also upregulates expression of anti-angiogenic genes including Thrombospondin-2. Furthermore, we show that while Hox A5 mRNA is expressed in quiescent endothelial cells (EC), its expression is diminished or absent in active angiogenic EC found in association with breast tumors or in proliferating infantile hemangiomas., Conclusions: Together our results suggest that restoring Hox A5 expression may provide a novel means to limit breast tumor growth or expansion of hemangiomas.

Angiogenesis is regulated by concerted actions of angiogenic and angiostatic factors. Homeobox D3 gene (HOXD3) is a potent proangiogenic transcription factor that promotes angiogenesis by modulating the expression of matrix-degrading proteinases, integrins, and extracellular matrix components. Application of HOXD3 can promote angiogenesis in the skin, but its role in other vascular beds has not been examined. The authors examined HOXD3 expression in human brain vessels by in situ hybridization. Although little or no HOXD3 mRNA was detected in normal brain vessels, increased levels of HOXD3 and its target gene, alpha V beta 3, were found in angiogenic vessels in human brain arteriovenous malformations. The authors further investigated whether HOXD3 plays a role in cerebral angiogenesis in a murine model. Expression of HOXD3 in mouse brain was achieved through retroviral vector-mediated HOXD3 gene transfer. HOXD3 expression lead to a significant induction of cerebral angiogenesis as shown by quantitative microvessel counting (HOXD3: 241 +/- 19 vessels/mm2 vs. saline: 150 +/- 14 vessels/mm2, P < 0.05). The data also showed that focal cerebral blood flow was increased in the angiogenic region with less vascular leakage. Moreover, expression of HOXD3 led to an increase in the expression of a direct downstream target gene alpha V beta 3 integrin. The data suggest that HOXD3 may play an important role in regulating cerebral angiogenesis, and that gene transfer of HOXD3 may provide a novel and potent means to stimulate angiogenesis.

Background: Cyclooxygenase (COX)-2-selective non-steroidal anti-inflammatory drugs have been used for anti-inflammatory therapy. However, it has also been described that they may increase risk of cardiovascular events., Objectives: To study the effects of COX2 inhibitor rofecoxib on platelet function using in vitro tests. Results were compared with those obtained in a parallel experiment with acetyl salicylic acid (ASA)., Methods: Studies of platelet aggregation, using different agonists, were performed by a turbidimetric method. Adhesive and cohesive function of platelets were analyzed by perfusion techniques, treated blood was exposed to thrombogenic surfaces and platelet interaction was morphometrically evaluated., Results: Twenty-five micro M of rofecoxib induced a prolonged lag time and a reduction in the percentage of aggregation when arachidonic acid, ADP or collagen were used as agonists. In perfusion studies with parallel chamber rofecoxib 50 microM and ASA 500 microM reduced overall platelet interaction with the collagen surface (17.4 +/- 3.7, P < 0.05; vs. 32.1 +/- 2.6%P < 0.05 and 17.9 +/- 2.4, vs. 31.9 +/- 3.24, P < 0.05, respectively). In studies performed on annular chambers, 25 micro M of rofecoxib reduced platelet interaction; values of the thrombus and covered surface were 17.4 +/- 4.5%; P < 0.05 and 21.1 +/- 4.1%; P < 0.05, respectively, vs. 30.4 +/- 7.5% and 33.5 +/- 6.5 in the control. ASA did also impair thrombus formation but differences did not reach the levels of statistical significance. Moreover, rofecoxib but not ASA reduced significantly thrombus height and thrombus area (7.4 +/- 0.5 microM; P < 0.005 and 96.0 +/- 21.2 microM(2); P < 0.05 vs. control 11.2 +/- 0.9 microM and 220.0 +/- 47.7 microM(2), respectively)., Conclusion: We conclude that under our experimental conditions, rofecoxib diminished platelet aggregation induced by different agonists and inhibited platelet-mediated thrombogenesis in an in vitro model of thrombosis.

Background: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets., Objective: To investigate signaling through tyrosine-phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets., Methods: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL(-1)) and type I collagen (Col-I; 20 microg mL(-1)), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col-I-coated coverslips, using citrated whole blood samples perfused through a parallel-plate chamber., Results and Conclusions: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.

Background and Objectives: Storage pool diseases (SPD) are heterogeneous disorders associated with an abnormal presence of intraplatelet granules, which cause mild to moderate bleeding diathesis. We investigated signaling through tyrosine phosphorylation of proteins occurring in platelets with total or partial absence of dense- and alpha-granules in response to activation., Design and Methods: We included a patient with severe delta-SPD, a patient with severe alpha-SPD or gray platelet syndrome, and six patients with partial deficiency of dense or a-granules. SPD was confirmed by electron microscopy evaluation of platelet ultrastructure. Platelet function was evaluated by bleeding time determination and conventional aggregometry. Platelet suspensions were activated with collagen and thrombin to analyze changes in tyrosine phosphorylation of proteins by electrophoresis and Western-blotting., Results: Bleeding times were prolonged in all the patients included. Aggregation responses were slightly decreased in delta-SPD and normal in the rest of patients. Tyrosine phosphorylation in platelets from patients with partial forms of SPD was equivalent to that observed in control platelets, absent in response to collagen and thrombin activation in delta-SPD, and deficient only to thrombin activation in alpha-SPD., Interpretation and Conclusions: Tyrosine phosphorylation of proteins in activated platelets is highly dependent on the substances contained in the dense-granules and moderately dependent on those contained in the alpha-granules. A minimum amount of intraplatelet granules ensures signaling through tyrosine phosphorylation of proteins.

After a vessel wall injury, platelets adhere to the subendothelium following a sequence of events: arrest of single platelets on the surface, progression to platelet spreading and final aggregation. Primary arrest of circulating platelets on subendothelial components occurs through platelet glycoprotein (GP) Ib and collagen receptors; then platelets spread and aggregate through a GPIIb-IIIa-dependent mechanism. A series of strategies were applied to analyse the tyrosine-phosphorylation mechanisms occurring at the different stages of platelet adhesion on subendothelial components under flow conditions, with special attention to primary arrest. To evaluate spread platelets, samples were exposed to acetylsalicylic acid, which blocks aggregate formation. To study single platelets in contact, a monoclonal antibody specific for GPIIb-IIIa was used to prevent platelet spreading and further aggregation. This experimental situation was also investigated using blood from two patients with Glanzmann's thrombasthenia (i.e. lacking GPIIb-IIIa). Results demonstrated that blockade of both spreading and aggregation results in significant changes in the tyrosine-phosphorylation patterns. Arrest of single platelets on collagen-rich surfaces resulted in phosphorylation of p125, identified as focal adhesion kinase (FAK), the 80/85 kDa doublet (cortactin), and p72, identified as Syk. Arrest of single platelets on von Willebrand factor as adhesive substrate showed that interaction through GPIb induces Syk phosphorylation, but not that of cortactin and FAK. Our data indicate that the initial arrest of platelets on subendothelial components involves Syk phosphorylation, which seems to be GPIb-dependent, and this is followed by activation and phosphorylation of cortactin and FAK. These processes seem to occur before GPIIb-IIIa becomes activated.

Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.