Esther Peña - Academia.edu (original) (raw)

Papers by Esther Peña

Research paper thumbnail of PAR2–SMAD3 in microvascular endothelial cells is indispensable for vascular stability via tissue factor signaling

Journal of Molecular Cell Biology, 2015

Tissue factor (TF) signaling regulates gene expression and protein synthesis leading to the modul... more 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.

Research paper thumbnail of 550 * ETS1/SMAD3 cooperation regulates TF-induced microvascular endothelial cell angiotube formation

Cardiovascular Research, 2014

ABSTRACT Purpose: Tissue factor (TF)-mediated signaling regulates gene expression and protein syn... more ABSTRACT Purpose: Tissue factor (TF)-mediated signaling regulates gene expression and protein synthesis leading to alterations in cell function. Recently we have demonstrated in microvascular endothelial cells (mECs) that TF induces ETS1, regulates CCL2 expression and stimulates smooth muscle cell recruitment stabilizing the newly formed microvessels. However, TF may signal through more than one transcription factor to regulate gene expression in motile mECs. We have investigated whether additional transcription factors are involved in mature microvessel formation. Methods: Human mECs were cultured in three dimensional basement membrane matrices. Cells were bioengineered to either abrogate or overexpress the TF gene. At 4 or 24 hours of culture RNA was obtained and real-time PCR gene arrays performed. Bioinformatic and systems biology analysis were applied to obtain the most probable regulated gene neural networks and targets. Targets genes were further validated. Results: TF activate different transcription factors as ETS1, KFL2, SMAD3, STAT3 and PPARγ to induce downstream expression of genes encoding molecules implicated in angiogenesis. SMAD3 through protein-protein interaction regulates ETS1 function. Whereas the ability of TF overexpressing cells to induce gene expression through ETS1 is dependent of AKT activation, in presence of SMAD3 ETS1 does not require AKT activation. SMAD3 bypasses the requirement of ETS1 for AKT activation and signals through ERK1/2 to activate target gene promoter activity. Conclusions: In mECs TF can trigger the expression of CCL2 through transcription factor synergism in a complex regulatory network with ETS1 as a main transcription factor to ensure the recruitment of perycites to stabilize newly formed microvessels.

Research paper thumbnail of Protein disulphide-isomerase A2 regulated intracellular tissue factor mobilisation in migrating human vascular smooth muscle cells

Thrombosis and haemostasis, Jan 30, 2015

Protein-disulphide isomerase family (PDI) are an ER-stress protein that controls TF-procoagulant ... more 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 fo...

Research paper thumbnail of 549 * Monocytes activate the non-canonical Wnt5a pathway in microvascular endothelial cells and induce tissue factor expression and tube formation

Cardiovascular Research, 2014

ABSTRACT Purpose: Atherosclerosis is characterized by lipid accumulation, inflammation and neovas... more ABSTRACT Purpose: Atherosclerosis is characterized by lipid accumulation, inflammation and neovascularization. Advanced atherosclerotic plaques can be destabilized and become vulnerable to rupture by microcapillary formation and inflammatory cell infiltration; and can lead to thrombosis and acute coronary syndromes. We have demonstrated in previous studies that increased tissue factor (TF) expression, that it is induced in microvascular endothelial cells (mECs), triggers angiogenesis; however, the signals that induce TF expression in mECs are barely understood. Here, we hypothesize that monocytes, that are strongly involved in atherosclerotic plaque inflammation, can interact with mECs and induce TF expression and, consequently, neovessel formation. Methods: The crosstalk between monocytes and mEC was studied in vitro by using both wound repair and matrigel assays. Angiogenesis was evaluated through tube length, nodule number and angiotube-covered area in the mEC cultured with MCM (isolated monocyte-derived conditioned medium). TF and Wnt5a genes were silenced through small interfering RNA (siRNA) in mEC and monocytes, TF expression was measured by PCR and immunoblotting and TF promoter was cloned into luciferase reporter vector pGL3. Finally, matrigel plugs seeded with monocytes and mECs were implanted in nude mice to test in vivo angiogenesis. Results: MCM enhanced wound repair, neovessel formation and expression of TF in in vitro assays. Wnt5a was identified as a protein released by monocytes that promoted TF expression and angiogenesis. Wnt5a protein increased up to 7 fold both TF expression in mEC and neotube formation. Furthermore, Wnt5a upregulated the receptor FZD5 in mEC and induced Ca2+ intracellular secretion and increased the transcription factor NF-κB for TF expression. In vivo, monocytes-mEC plugs showed significantly higher angiogenesis. Conclusions: This study demonstrate that monocytes crosstalk with mEC through Wnt5a secreted monocyte activates the non-canonical Wnt-pathway in mEC by interacting with FZD5 and triggering TF expression and inducing angiogenesis.

Research paper thumbnail of Monocyte-secreted Wnt5a interacts with FZD5 in microvascular endothelial cells and induces angiogenesis through tissue factor signaling

Journal of molecular cell biology, 2014

Angiogenesis during reactive and pathologic processes is characteristically associated with infla... more 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...

Research paper thumbnail of Tissue factor-Akt signaling triggers microvessel formation

Journal of Thrombosis and Haemostasis, 2012

Research paper thumbnail of Tissue Factor Induces Human Coronary Artery Smooth Muscle Cell Motility through Wnt-Signalling

Journal of Thrombosis and Haemostasis, 2013

Tissue factor (TF) is the most relevant physiological trigger of thrombosis contributing to the p... more 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. 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. 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. 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. 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.

Research paper thumbnail of Subcellular localization of tissue factor and human coronary artery smooth muscle cell migration

Journal of Thrombosis and Haemostasis, 2012

Background: Tissue factor (TF) is the most relevant physiological trigger of thrombosis. Addition... more 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.

Research paper thumbnail of Tissue Factor Regulates Microvessel Formation and Stabilization by Induction of Chemokine (C-C motif) Ligand 2 Expression

Arteriosclerosis, Thrombosis, and Vascular Biology, 2011

Objective-Tissue factor (TF) triggers arterial thrombosis. TF is also able to initiate cellular s... more 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.

Research paper thumbnail of Ets-1 transcription is required in tissue factor driven microvessel formation and stabilization

Angiogenesis, 2012

Tissue factor (TF) has well-recognized roles as initiator of blood coagulation as well as an intr... more 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.

Research paper thumbnail of Angiogenic microvascular endothelial cells release microparticles rich in tissue factor that promotes postischemic collateral vessel formation

Arteriosclerosis, thrombosis, and vascular biology, 2015

Therapeutic angiogenesis is a promising strategy for treating ischemia. Our previous work showed ... more 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. 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...

Research paper thumbnail of PAR2–SMAD3 in microvascular endothelial cells is indispensable for vascular stability via tissue factor signaling

Journal of Molecular Cell Biology, 2015

Tissue factor (TF) signaling regulates gene expression and protein synthesis leading to the modul... more 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.

Research paper thumbnail of 550 * ETS1/SMAD3 cooperation regulates TF-induced microvascular endothelial cell angiotube formation

Cardiovascular Research, 2014

ABSTRACT Purpose: Tissue factor (TF)-mediated signaling regulates gene expression and protein syn... more ABSTRACT Purpose: Tissue factor (TF)-mediated signaling regulates gene expression and protein synthesis leading to alterations in cell function. Recently we have demonstrated in microvascular endothelial cells (mECs) that TF induces ETS1, regulates CCL2 expression and stimulates smooth muscle cell recruitment stabilizing the newly formed microvessels. However, TF may signal through more than one transcription factor to regulate gene expression in motile mECs. We have investigated whether additional transcription factors are involved in mature microvessel formation. Methods: Human mECs were cultured in three dimensional basement membrane matrices. Cells were bioengineered to either abrogate or overexpress the TF gene. At 4 or 24 hours of culture RNA was obtained and real-time PCR gene arrays performed. Bioinformatic and systems biology analysis were applied to obtain the most probable regulated gene neural networks and targets. Targets genes were further validated. Results: TF activate different transcription factors as ETS1, KFL2, SMAD3, STAT3 and PPARγ to induce downstream expression of genes encoding molecules implicated in angiogenesis. SMAD3 through protein-protein interaction regulates ETS1 function. Whereas the ability of TF overexpressing cells to induce gene expression through ETS1 is dependent of AKT activation, in presence of SMAD3 ETS1 does not require AKT activation. SMAD3 bypasses the requirement of ETS1 for AKT activation and signals through ERK1/2 to activate target gene promoter activity. Conclusions: In mECs TF can trigger the expression of CCL2 through transcription factor synergism in a complex regulatory network with ETS1 as a main transcription factor to ensure the recruitment of perycites to stabilize newly formed microvessels.

Research paper thumbnail of Protein disulphide-isomerase A2 regulated intracellular tissue factor mobilisation in migrating human vascular smooth muscle cells

Thrombosis and haemostasis, Jan 30, 2015

Protein-disulphide isomerase family (PDI) are an ER-stress protein that controls TF-procoagulant ... more 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 fo...

Research paper thumbnail of 549 * Monocytes activate the non-canonical Wnt5a pathway in microvascular endothelial cells and induce tissue factor expression and tube formation

Cardiovascular Research, 2014

ABSTRACT Purpose: Atherosclerosis is characterized by lipid accumulation, inflammation and neovas... more ABSTRACT Purpose: Atherosclerosis is characterized by lipid accumulation, inflammation and neovascularization. Advanced atherosclerotic plaques can be destabilized and become vulnerable to rupture by microcapillary formation and inflammatory cell infiltration; and can lead to thrombosis and acute coronary syndromes. We have demonstrated in previous studies that increased tissue factor (TF) expression, that it is induced in microvascular endothelial cells (mECs), triggers angiogenesis; however, the signals that induce TF expression in mECs are barely understood. Here, we hypothesize that monocytes, that are strongly involved in atherosclerotic plaque inflammation, can interact with mECs and induce TF expression and, consequently, neovessel formation. Methods: The crosstalk between monocytes and mEC was studied in vitro by using both wound repair and matrigel assays. Angiogenesis was evaluated through tube length, nodule number and angiotube-covered area in the mEC cultured with MCM (isolated monocyte-derived conditioned medium). TF and Wnt5a genes were silenced through small interfering RNA (siRNA) in mEC and monocytes, TF expression was measured by PCR and immunoblotting and TF promoter was cloned into luciferase reporter vector pGL3. Finally, matrigel plugs seeded with monocytes and mECs were implanted in nude mice to test in vivo angiogenesis. Results: MCM enhanced wound repair, neovessel formation and expression of TF in in vitro assays. Wnt5a was identified as a protein released by monocytes that promoted TF expression and angiogenesis. Wnt5a protein increased up to 7 fold both TF expression in mEC and neotube formation. Furthermore, Wnt5a upregulated the receptor FZD5 in mEC and induced Ca2+ intracellular secretion and increased the transcription factor NF-κB for TF expression. In vivo, monocytes-mEC plugs showed significantly higher angiogenesis. Conclusions: This study demonstrate that monocytes crosstalk with mEC through Wnt5a secreted monocyte activates the non-canonical Wnt-pathway in mEC by interacting with FZD5 and triggering TF expression and inducing angiogenesis.

Research paper thumbnail of Monocyte-secreted Wnt5a interacts with FZD5 in microvascular endothelial cells and induces angiogenesis through tissue factor signaling

Journal of molecular cell biology, 2014

Angiogenesis during reactive and pathologic processes is characteristically associated with infla... more 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...

Research paper thumbnail of Tissue factor-Akt signaling triggers microvessel formation

Journal of Thrombosis and Haemostasis, 2012

Research paper thumbnail of Tissue Factor Induces Human Coronary Artery Smooth Muscle Cell Motility through Wnt-Signalling

Journal of Thrombosis and Haemostasis, 2013

Tissue factor (TF) is the most relevant physiological trigger of thrombosis contributing to the p... more 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. 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. 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. 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. 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.

Research paper thumbnail of Subcellular localization of tissue factor and human coronary artery smooth muscle cell migration

Journal of Thrombosis and Haemostasis, 2012

Background: Tissue factor (TF) is the most relevant physiological trigger of thrombosis. Addition... more 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.

Research paper thumbnail of Tissue Factor Regulates Microvessel Formation and Stabilization by Induction of Chemokine (C-C motif) Ligand 2 Expression

Arteriosclerosis, Thrombosis, and Vascular Biology, 2011

Objective-Tissue factor (TF) triggers arterial thrombosis. TF is also able to initiate cellular s... more 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.

Research paper thumbnail of Ets-1 transcription is required in tissue factor driven microvessel formation and stabilization

Angiogenesis, 2012

Tissue factor (TF) has well-recognized roles as initiator of blood coagulation as well as an intr... more 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.

Research paper thumbnail of Angiogenic microvascular endothelial cells release microparticles rich in tissue factor that promotes postischemic collateral vessel formation

Arteriosclerosis, thrombosis, and vascular biology, 2015

Therapeutic angiogenesis is a promising strategy for treating ischemia. Our previous work showed ... more 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. 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...