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Massimo Santoro - One of the best experts on this subject based on the ideXlab platform.
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Cilia Control Vascular Mural Cell Recruitment in Vertebrates
Cell reports, 2017Co-Authors: Xiaowen Chen, Dafne Gays, Carlo Milia, Massimo SantoroAbstract:Vascular Mural Cells (vMCs) are essential components of the vertebrate vascular system, controlling blood vessel maturation and homeostasis. Discrete molecular mechanisms have been associated with vMC development and differentiation. The function of hemodynamic forces in controlling vMC recruitment is unclear. Using transgenic lines marking developing vMCs in zebrafish embryos, we find that vMCs are recruited by arterial-fated vessels and that the process is flow dependent. We take advantage of tissue-specific CRISPR gene targeting to demonstrate that hemodynamic-dependent Notch activation and the ensuing arterial genetic program is driven by endothelial primary cilia. We also identify zebrafish foxc1b as a cilia-dependent Notch-specific target that is required within endothelial Cells to drive vMC recruitment. In summary, we have identified a hemodynamic-dependent mechanism in the developing vasculature that controls vMC recruitment.
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Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons
Cell reports, 2015Co-Authors: Vitor Fortuna, Massimo Santoro, Luc Pardanaud, Isabelle Brunet, Roxana Ola, Emma Ristori, Stefania Nicoli, Anne EichmannAbstract:Summary The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular Mural Cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial Cells but have VMCs. Thus, PDGFR-mediated Mural Cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.
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an α smooth muscle actin acta2 αsma zebrafish transgenic line marking vascular Mural Cells and visceral smooth muscle Cells
PLOS ONE, 2014Co-Authors: Thomas Whitesell, Massimo Santoro, Regan M Kennedy, Alyson D Carter, Evvilynn Rollins, Sonja Georgijevic, Sarah J ChildsAbstract:Mural Cells of the vascular system include vascular smooth muscle Cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of Mural Cell development in vertebrates is α smooth muscle actin (acta2; αsma), which is expressed by pericytes and SMCs. In vivo models of vascular Mural Cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing Cells. These transgenic fish were used to trace the live development of Mural Cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive Cells and the number of types of blood vessels associated with Mural Cells increases. Interestingly, the Mural Cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish Mural Cells develop relatively late, and have little mobility once they associate with vessels.
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An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular Mural Cells and visceral smooth muscle Cells.
PloS one, 2014Co-Authors: Thomas Whitesell, Massimo Santoro, Regan M Kennedy, Alyson D Carter, Evvilynn Rollins, Sonja Georgijevic, Sarah J ChildsAbstract:Mural Cells of the vascular system include vascular smooth muscle Cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of Mural Cell development in vertebrates is α smooth muscle actin (acta2; αsma), which is expressed by pericytes and SMCs. In vivo models of vascular Mural Cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing Cells. These transgenic fish were used to trace the live development of Mural Cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive Cells and the number of types of blood vessels associated with Mural Cells increases. Interestingly, the Mural Cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish Mural Cells develop relatively late, and have little mobility once they associate with vessels.
Sarah J Childs - One of the best experts on this subject based on the ideXlab platform.
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an α smooth muscle actin acta2 αsma zebrafish transgenic line marking vascular Mural Cells and visceral smooth muscle Cells
PLOS ONE, 2014Co-Authors: Thomas Whitesell, Massimo Santoro, Regan M Kennedy, Alyson D Carter, Evvilynn Rollins, Sonja Georgijevic, Sarah J ChildsAbstract:Mural Cells of the vascular system include vascular smooth muscle Cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of Mural Cell development in vertebrates is α smooth muscle actin (acta2; αsma), which is expressed by pericytes and SMCs. In vivo models of vascular Mural Cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing Cells. These transgenic fish were used to trace the live development of Mural Cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive Cells and the number of types of blood vessels associated with Mural Cells increases. Interestingly, the Mural Cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish Mural Cells develop relatively late, and have little mobility once they associate with vessels.
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An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular Mural Cells and visceral smooth muscle Cells.
PloS one, 2014Co-Authors: Thomas Whitesell, Massimo Santoro, Regan M Kennedy, Alyson D Carter, Evvilynn Rollins, Sonja Georgijevic, Sarah J ChildsAbstract:Mural Cells of the vascular system include vascular smooth muscle Cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of Mural Cell development in vertebrates is α smooth muscle actin (acta2; αsma), which is expressed by pericytes and SMCs. In vivo models of vascular Mural Cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing Cells. These transgenic fish were used to trace the live development of Mural Cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive Cells and the number of types of blood vessels associated with Mural Cells increases. Interestingly, the Mural Cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish Mural Cells develop relatively late, and have little mobility once they associate with vessels.
Wanqiu Chen - One of the best experts on this subject based on the ideXlab platform.
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abstract tmp111 thalidomide and lenalidomide treatment reduces microhemorrhage in brain arteriovenous malformation in mice through increasing Mural Cell coverage
Stroke, 2017Co-Authors: Wan Zhu, Wanqiu Chen, Rui Zhang, Dingquan Zou, Chen Bao, Lei Zhan, Meng Zhang, Ethan A Winkler, Michael T LawtonAbstract:Introduction: Brain arteriovenous malformations (bAVMs) have an abnormal vessel wall and are prone to rupture. The mechanism of bAVM rupture is unclear. In Alk1 -deficient mice, bAVM vessels have fewer Mural Cells. In endoglin-deficient mice, thalidomide increases Mural Cells in retina AVM vessels. We hypothesize that thalidomide and its less toxic analogue, lenalidomide, improve vessel Mural Cell coverage and reduce microhemorrhage in Alk1 -deficient bAVM. Methods: Brain AVMs were induced in adult Alk1 2f/2f mice through induction of focal Alk1 gene deletion and angiogenic stimulation. Thalidomide was injected intraperitoneally (i.p.) twice per week for six weeks, starting either 2 weeks after model induction when bAVMs were beginning to develop or 8 weeks after when bAVMs were fully developed. Lenalidomide treatment was started 8 weeks after model induction through i.p. injection daily for six weeks. Results: Thalidomide treatment starting 2 weeks after bAVM induction reduced the number of abnormal vessels and microhemorrhage and increased vascular smooth muscle (vSM)-coverage. Thalidomide also increased the expression of platelet-derived growth factor b (pdgfb) and its receptor (pdgfr beta), indicating that pdgfg/pdgfr beta signaling is one of the mechanisms responsible for the improvement of Mural Cell coverage. Thalidomide and lenalidomide treatment started at the later time point also improved vSM-coverage and showed a trend toward reduction of microhemorrhage and abnormal vessel count. Conclusions: Thalidomide and lenalidomide stabilize the bAVM vessel wall and reduce microhemorrahge. Further studies are needed to determine whether these agents have a possible therapeutic value for patients.
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reduced Mural Cell coverage and impaired vessel integrity after angiogenic stimulation in the alk1 deficient brain
Arteriosclerosis Thrombosis and Vascular Biology, 2013Co-Authors: Wanqiu Chen, Kristine Jun, Dimitrios Davalos, Fanxia Shen, Espen J Walker, Vincent Degos, Tarik Tihan, Yi Guo, Michael T Lawton, Katerina AkassoglouAbstract:Objective— Vessels in brain arteriovenous malformations are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 patients with activin receptor-like kinase 1 ( Alk1 ) mutation have a higher incidence of brain arteriovenous malformation than the general population. We tested the hypothesis that vascular endothelial growth factor impairs vascular integrity in the Alk1 -deficient brain through reduction of Mural Cell coverage. Methods and Results— Adult Alk1 1f/2f mice (loxP sites flanking exons 4–6) and wild-type mice were injected with 2×107 PFU adenovious-cre recombinase and 2×109 genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk1 1f/2f mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1 -deficient brain had more fibrin (99±30×103 pixels/mm2 versus 40±13×103; P =0.001), iron deposition (508±506 pixels/mm2 versus 6±49; P =0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ Cells/mm2 versus 240±104 Iba1+; P =0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1 -deficient brain had more α-smooth muscle actin negative vessels (52±9% versus 12±7%, P <0.001), fewer vascular-associated pericytes (503±179/mm2 versus 931±115, P <0.001), and reduced platelet-derived growth factor receptor-β expression. Conclusion— Reduction of Mural Cell coverage in response to vascular endothelial growth factor stimulation is a potential mechanism for the impairment of vessel wall integrity in hereditary hemorrhagic telangiectasia type 2-associated brain arteriovenous malformation.
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Abstract TMP118: Alk1 Deficiency Impairs Mural Cell Recruitment During Brain Angiogenesis
Stroke, 2013Co-Authors: Wanqiu Chen, Kristine Jun, Yi Guo, Mamta Wankhede, William L. YoungAbstract:Background and Purpose: VEGF stimulation induced dysplasia vessel formation in the Alk1 -deficient brain. The dysplastic vessels had less Mural Cell coverage associated with increased extravasation of blood contents. Platelet-derived growth factor B (PDGFB) signaling is important in Mural Cell recruitment during angiogenesis. Notch signaling is upstream of PDGFB. We hypothesized that Alk1 deletion impairs pericyte recruitment through downregulation of DLL4/PDGFB signaling. Thalidomide can increase PDGFB expression and was used to test our hypothesis in vivo. Methods: Human brain microvascular endothelial Cells (HBMECs) were infected with a lentiviral vector carrying ALK1 shRNA. Cells with 80% reduction of ALK1 gene expression were treated with vascular endothelial growth factor (VEGF; 0, 10, 50, and 100 ng/ml) for 18 hours. DLL4 and PDGFB mRNA were measured using real-time PCR. Pericyte-recruitment was evaluated by co-culturing pericytes with HBMECs. Cerebrovascular dysplasia was induced by co-injection of an adenoviral vector expressing cre-recombinase and an adeno-associated viral vector expressing VEGF into the basal ganglia of Alk1 -floxed mice. Two weeks later, thalidomide (75 mg/kg, i.p.) or DMSO was administered twice per week for six weeks. Results: Knockdown of ALK1 attenuated the increase of DLL4 and PDGFB in HBMECs following VEGF stimulation. DLL4 and PDGFB expression was highly correlated (R 2 = 0.93). ALK1 knockdown reduced the ability of HBMEC to recruit pericytes (P = 0.014). In vivo, thalidomide treatment significantly reduced vascular dysplasia (dysplastic vessels: thalidomide vs. DMSO, 3.4±1.2/200 vessels vs. 1.0±0.5, P = 0.003), without affecting vessel density (P = 0.11). Smooth muscle-negative dysplastic vessels decreased in thalidomide-treated mice, compared to DMSO-treated mice (21%±6 vs 47±9, P = 0.0002). Prussian blue-positive area was reduced in the thalidomide group, compared to the DMSO group (P = 0.0011). Conclusions: Our findings suggest that Alk1 regulates pericyte recruitment through DLL4/PDGFB signaling during brain angiogenesis.
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Reduced Mural Cell coverage and impaired vessel integrity after angiogenic stimulation in the Alk1-deficient brain
Arteriosclerosis Thrombosis and Vascular Biology, 2013Co-Authors: Wanqiu Chen, Kristine Jun, S. Paul Oh, Fanxia Shen, Espen J Walker, Vincent Degos, Tarik Tihan, Yi Guo, Michael T Lawton, Dimitrios DavalosAbstract:OBJECTIVE: Vessels in brain arteriovenous malformations are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of brain arteriovenous malformation than the general population. We tested the hypothesis that vascular endothelial growth factor impairs vascular integrity in the Alk1-deficient brain through reduction of Mural Cell coverage.\n\nMETHODS AND RESULTS: Adult Alk1(1f/2f) mice (loxP sites flanking exons 4-6) and wild-type mice were injected with 2×10(7) PFU adenovious-cre recombinase and 2×10(9) genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk1(1f/2f) mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1-deficient brain had more fibrin (99±30×10(3) pixels/mm(2) versus 40±13×10(3); P=0.001), iron deposition (508±506 pixels/mm(2) versus 6±49; P=0.04), and Iba1(+) microglia/macrophage infiltration (888±420 Iba1(+) Cells/mm(2) versus 240±104 Iba1(+); P=0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-smooth muscle actin negative vessels (52±9% versus 12±7%, P
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abstract 3169 reduced pdgfr beta expression after regional alk1 deletion and vegf stimulation in the brain is associated with reduced Mural Cell coverage
Stroke, 2012Co-Authors: Wanqiu Chen, Yi Guo, Andrew W Bollen, William L. YoungAbstract:Background and Purpose: About 2% of brain arteriovenous malformations (bAVM) are attributable to Hereditary Hemorrhagic Telangectasia (HHT). Previously we created a bAVM model in the adult mouse using regional Alk1 (the causal gene in HHT2) deletion and VEGF stimulation, and reported that the dysplastic vessels in this model have less smooth muscle Cell coverage than the normal cerebrovasculature. We hypothesized that VEGF stimulation in the setting of Alk1 loss-of-function leads to impaired Mural Cell coverage through reduction of platelet-derived growth factor receptor (PDGFR) signaling, and is associated with impaired cerebrovascular integrity. Methods: Brain AVM formation was induced by injection of 2X107 PFU adenoviral vector expressing cre-recombinase (AdCre) and 2X109 genome copies of adeno-associated viral vector (AAV) expressing VEGF (AAV-VEGF) into the basal ganglia of Alk1-floxed mice (loxP sites flanking Exons 4-6). The brain samples were collected 8 weeks after vector injection. Intra-parenchymal hemorrhage, microglial/macrophage counts, Alk1 and PDGFR/PDGFB protein expression were evaluated using Prussian blue staining, Iba1 staining and Western blot. Results: We found a 66±27% reduction in Alk1 protein expression around the injection site of the AdCre and AAV-VEGF-treated group compared with the control vector treated group (p=0.002, ANOVA). Fresh red blood Cells (RBCs) were detected in the extravascular space in the Alk1-deficient brain parenchyma (Ad-Cre-treated Alk1-floxed mice), with ≈10-fold increase of Prussian blue coverage area compared to the brain with intact Alk1 gene (Ad-GFP-treated Alk1-floxed mice; p=0.016, rank sum test; or AdCre-treated wild-type mice, p=0.010, rank sum test) after VEGF stimulation. The area of Prussian blue coverage was positively correlated with the number of Iba1+ Cells (R2=0.39, p Conclusions: These results indicate an important role for Alk1 in maintaining cerebrovascular integrity as shown by its effect on pericyte function through interaction with PDGFR-beta signaling. Alk1 deletion plus VEGF stimulation reduced vessel wall integrity that was associated with an inflammatory response. Altered pericyte-endothelial Cell interaction may play a causal role in the pathogenesis of human bAVM, possibly involving a local inflammatory response associated with RBC extravasation.
Karen K Hirschi - One of the best experts on this subject based on the ideXlab platform.
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Connexin45 Regulates Endothelial-Induced Mesenchymal Cell Differentiation Toward a Mural Cell Phenotype
Arteriosclerosis thrombosis and vascular biology, 2012Co-Authors: Jennifer S. Fang, Cuiping Dai, David T. Kurjiaka, Janis M. Burt, Karen K HirschiAbstract:Objective— The focus of this study was to investigate the role of connexin (Cx) 45 in endothelial-induced Mural Cell differentiation. Methods and Results— We created Mural Cell precursors that stably express only Cx45 in Cx43-deficient mesenchymal Cells (ReCx45), and used our in vitro model of blood vessel assembly to assess the capacity of this Cx to support endothelial-induced Mural Cell differentiation. Lucifer Yellow dye injection and dual whole-Cell patch clamping revealed that functional gap junctions exhibiting properties of Cx45-containing channels formed among ReCx45 transfectants, and between ReCx45 and endothelial Cells. HeteroCellular Cx45-containing gap junction channels enabled transforming growth factor-β activation and promoted the upregulation of Mural Cell–specific proteins in the mesenchymal precursors. Conclusion— These studies reveal a critical role for Cx45 in the regulation of endothelial-induced Mural Cell differentiation, which is consistent with the phenotype of Cx45-deficient embryos that exhibit dysregulated transforming growth factor-β and lack Mural Cell development.
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Gap Junction Communication Mediates Transforming Growth Factor-β Activation and Endothelial-Induced Mural Cell Differentiation
Circulation research, 2003Co-Authors: Karen K Hirschi, Janis M. Burt, Kendal D. Hirschi, Cuiping DaiAbstract:During blood vessel assembly, endothelial Cells recruit mesenchymal progenitors and induce their differentiation into Mural Cells via contact-dependent transforming growth factor-β (TGF-β) activation. We investigated whether gap junction channels are formed between endothelial Cells and recruited mesenchymal progenitors and whether interCellular communication is necessary for endothelial-induced Mural Cell differentiation. Mesenchymal progenitors from Cx43 −/− murine embryos and Cx43 +/+ littermates were cocultured with prelabeled endothelial Cells. IntraCellular dye injection and dual whole-Cell voltage clamp revealed that endothelial Cells formed gap junction channels with Cx43 +/+ but not Cx43 −/− progenitors. In coculture with endothelial Cells, Cx43 −/− progenitors did not undergo Mural Cell differentiation as did Cx43 +/+ Cells. Stable reexpression of Cx43 in Cx43 −/− Cells (reCx43) restored their ability to form gap junctions with endothelial Cells and undergo endothelial-induced Mural Cell differentiation. Cocultures of endothelial Cells and either Cx43 +/+ or reCx43 mesenchymal Cells produced activated TGF-β; endothelial-Cx43 −/− cocultures did not. However, Cx43 −/− Cells did produce latent TGF-β and undergo Mural Cell differentiation in response to exogenous TGF-β1. These studies indicate that gap junction communication between endothelial and mesenchymal Cells mediates TGF-β activation and subsequent Mural Cell differentiation.
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endothelial Cells modulate the proliferation of Mural Cell precursors via platelet derived growth factor bb and heterotypic Cell contact
Circulation Research, 1999Co-Authors: Karen K Hirschi, Stephanie Rohovsky, Laurence H Beck, Sandra Smith, Patricia A DamoreAbstract:Abstract —Embryological data suggest that endothelial Cells (ECs) direct the recruitment and differentiation of Mural Cell precursors. We have developed in vitro coculture systems to model some of these events and have shown that ECs direct the migration of undifferentiated mesenchymal Cells (10T1/2 Cells) and induce their differentiation toward a smooth muscle Cell/pericyte lineage. The present study was undertaken to investigate Cell proliferation in these cocultures. ECs and 10T1/2 Cells were cocultured in an underagarose assay in the absence of contact. There was a 2-fold increase in bromodeoxyuridine labeling of 10T1/2 Cells in response to ECs, which was completely inhibited by the inclusion of neutralizing antiserum against platelet-derived growth factor (PDGF)-B. Antisera against PDGF-A, basic fibroblast growth factor, or transforming growth factor (TGF)-β had no effect on EC-stimulated 10T1/2 Cell proliferation. EC proliferation was not influenced by coculture with 10T1/2 Cells in the absence of contact. The Cells were then cocultured so that contact was permitted. Double labeling and fluorescence-activated Cell sorter analysis revealed that ECs and 10T1/2 Cells were growth-inhibited by 43% and 47%, respectively. Conditioned media from contacting EC-10T1/2 Cell cocultures inhibited the growth of both Cell types by 61% and 48%, respectively. Although we have previously shown a role for TGF-β in coculture-induced Mural Cell differentiation, growth inhibition resulting from contacting cocultures or conditioned media was not suppressed by the presence of neutralizing antiserum against TGF-β. Furthermore, the decreased proliferation of 10T1/2 Cells in the direct cocultures could not be attributed to downregulation of the PDGF-B in ECs or the PDGF receptor-β in the 10T1/2 Cells. Our data suggest that modulation of proliferation occurs during EC recruitment of mesenchymal Cells and that heterotypic Cell-Cell contact and soluble factors play a role in growth control during vessel assembly.
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Endothelial Cells Modulate the Proliferation of Mural Cell Precursors via Platelet-Derived Growth Factor-BB and Heterotypic Cell Contact
Circulation research, 1999Co-Authors: Karen K Hirschi, Stephanie Rohovsky, Laurence H Beck, Sandra R. Smith, Patricia A. D'amoreAbstract:Embryological data suggest that endothelial Cells (ECs) direct the recruitment and differentiation of Mural Cell precursors. We have developed in vitro coculture systems to model some of these events and have shown that ECs direct the migration of undifferentiated mesenchymal Cells (10T1/2 Cells) and induce their differentiation toward a smooth muscle Cell/pericyte lineage. The present study was undertaken to investigate Cell proliferation in these cocultures. ECs and 10T1/2 Cells were cocultured in an underagarose assay in the absence of contact. There was a 2-fold increase in bromodeoxyuridine labeling of 10T1/2 Cells in response to ECs, which was completely inhibited by the inclusion of neutralizing antiserum against platelet-derived growth factor (PDGF)-B. Antisera against PDGF-A, basic fibroblast growth factor, or transforming growth factor (TGF)-beta had no effect on EC-stimulated 10T1/2 Cell proliferation. EC proliferation was not influenced by coculture with 10T1/2 Cells in the absence of contact. The Cells were then cocultured so that contact was permitted. Double labeling and fluorescence-activated Cell sorter analysis revealed that ECs and 10T1/2 Cells were growth-inhibited by 43% and 47%, respectively. Conditioned media from contacting EC-10T1/2 Cell cocultures inhibited the growth of both Cell types by 61% and 48%, respectively. Although we have previously shown a role for TGF-beta in coculture-induced Mural Cell differentiation, growth inhibition resulting from contacting cocultures or conditioned media was not suppressed by the presence of neutralizing antiserum against TGF-beta. Furthermore, the decreased proliferation of 10T1/2 Cells in the direct cocultures could not be attributed to downregulation of the PDGF-B in ECs or the PDGF receptor-beta in the 10T1/2 Cells. Our data suggest that modulation of proliferation occurs during EC recruitment of mesenchymal Cells and that heterotypic Cell-Cell contact and soluble factors play a role in growth control during vessel assembly.
Roberto F. Nicosia - One of the best experts on this subject based on the ideXlab platform.
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Regulation of angiogenesis, Mural Cell recruitment and adventitial macrophage behavior by Toll-like receptors
Angiogenesis, 2013Co-Authors: Alfred C. Aplin, Eric Fogel, Giovanni Ligresti, Penelope Zorzi, Kelly D. Smith, Roberto F. NicosiaAbstract:The angiogenic response to injury can be studied by culturing rat or mouse aortic explants in collagen gels. Gene expression studies show that aortic angiogenesis is preceded by an immune reaction with overexpression of Toll-like receptors (TLRs) and TLR-inducible genes. TLR1, 3, and 6 are transiently upregulated at 24 h whereas TLR2, 4, and 8 expression peaks at 24 h but remains elevated during angiogenesis and vascular regression. Expression of TLR5, 7 and 9 steadily increases over time and is highest during vascular regression. Studies with isolated Cells show that TLRs are expressed at higher levels in aortic macrophages compared to endothelial or Mural Cells with the exception of TLR2 and TLR9 which are more abundant in the aortic endothelium. LPS and other TLR ligands dose dependently stimulate angiogenesis and vascular endothelial growth factor production. TLR9 ligands also influence the behavior of nonendothelial Cell types by blocking Mural Cell recruitment and inducing formation of multinucleated giant Cells by macrophages. TLR9-induced Mural Cell depletion is associated with reduced expression of the Mural Cell recruiting factor PDGFB. The spontaneous angiogenic response of the aortic rings to injury is reduced in cultures from mice deficient in myeloid differentiation primary response 88 (MyD88), a key adapter molecule of TLRs, and following treatment with an inhibitor of the NFκB pathway. These results suggest that the TLR system participates in the angiogenic response of the vessel wall to injury and may play an important role in the regulation of inflammatory angiogenesis in reactive and pathologic processes.
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MCP-1 promotes Mural Cell recruitment during angiogenesis in the aortic ring model
Angiogenesis, 2010Co-Authors: Alfred C. Aplin, Eric Fogel, Roberto F. NicosiaAbstract:Rings of rat or mouse aorta embedded in collagen gels produce angiogenic outgrowths in response to the injury of the dissection procedure. Aortic outgrowths are composed of branching endothelial tubes and surrounding Mural Cells. Mural Cells emerge following endothelial sprouting and gradually increase during the maturation of the neovessels. Treatment of aortic cultures with angiopoietin-1 (Ang-1), an angiogenic factor implicated in vascular maturation and remodeling, stimulates the Mural Cell recruitment process. Ang-1 induces expression of many cytokines and chemokines including monocyte chemotactic protein-1 (MCP-1). Inhibition of p38 MAP kinase, a signaling molecule required for Mural Cell recruitment, blocks Ang1-induced MCP-1 expression. Recombinant MCP-1 dose-dependently increases Mural Cell number while an anti-MCP-1 blocking antibody reduces it. In addition, antibody mediated neutralization of MCP-1 abrogates the stimulatory effect of Ang-1 on Mural Cell recruitment. Aortic rings from genetically modified mice deficient in MCP-1 or its receptor CCR2 have fewer Mural Cells than controls. MCP-1 deficiency also impairs the Mural Cell recruitment activity of Ang-1. Our studies indicate that spontaneous and Ang1-induced Mural Cell recruitment in the aortic ring of model of angiogenesis are in part mediated by MCP-1. These results implicate MCP-1 as one of the mediators of Mural Cell recruitment in the aortic ring model, and suggest that chemokine pathways may contribute to the assembly of the vessel wall during the angiogenesis response to injury.
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rat aorta derived Mural precursor Cells express the tie2 receptor and respond directly to stimulation by angiopoietins
Journal of Cell Science, 2003Co-Authors: Monica Iurlaro, Eric Fogel, Marta Scatena, Susan L Wieting, Roberto F. NicosiaAbstract:Recent studies have implicated the Tie2 tyrosine-kinase receptor and its main ligands - angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) - as crucial regulators of Mural Cell recruitment during angiogenesis. Angiopoietin-mediated activation of Tie2 promotes perivascular Mural Cell assembly, but the mechanisms regulating this process are poorly understood because differentiated Mural Cells do not have the Tie2 receptor, which is reportedly expressed only in endothelial Cells. There is also no direct evidence that Tie2 activation results in production of Mural Cell chemoattractants by the endothelium. In the rat aorta model of angiogenesis, developing microvessels recruit Mural Cells from the intimal/subintimal layers of the aortic wall. Ang-1 and Ang-2 promote angiogenesis in this system, stimulating branching morphogenesis and Mural Cell assembly. Mural precursor Cells (MPCs) isolated with a nonenzymatic method from the intimal aspect of the rat aorta were positive for smooth muscle Cell markers (α-smooth muscle actin and calponin) and negative for endothelial markers (factor-VIII-related antigen and CD31). These Cells responded chemotactically to Ang-1 and Ang-2, and secreted MMP-2 when treated with these factors. Western-blot analysis, immunocytochemistry and RT-PCR demonstrated that MPCs express the Tie2 receptor. Immunoprecipitation showed phosphorylation of MPC Tie2 on tyrosine residues upon stimulation with Ang-1 or Ang-2. Surface expression of Tie2 was further demonstrated by isolating Tie2 + /α-smooth muscle actin + MPCs from primary aortic outgrowths with anti-Tie2-IgG-coated magnetic beads. Immunostaining of the rat aorta confirmed expression of Tie2 not only in endothelial Cells but also in nonendothelial mesenchymal Cells located in the aortic intimal/subintimal layers, which are the source of MPCs. These data indicate that the aortic wall contains Tie2 + nonendothelial mesenchymal Cells and suggest that Tie2-related recruitment of Mural Cells during angiogenesis may occur through angiopoietin-mediated direct stimulation of these Cells.
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Requisite role of p38 MAPK in Mural Cell recruitment during angiogenesis in the rat aorta model.
Journal of vascular research, 2003Co-Authors: Wen Hui Zhu, Jiahuai Han, Roberto F. NicosiaAbstract:During the early stage of angiogenesis, neovascular sprouts are composed primarily of endothelial Cells. As they mature, microvessels acquire a coating of Mural Cells, which are critical for the development and maintenance of a functional vasculature. Though growth factor regulation of Mural Cell recruitment has been extensively investigated, the intraCellular signaling events involved in this process remain poorly understood. Among the intraCellular kinases implicated in angiogenesis, the p38 MAPK has been shown to transduce signals critical for vascular remodeling and maturation. The rat aorta model of angiogenesis was used to further investigate the role of this signaling pathway in the recruitment of Mural Cells during angiogenesis. The p38 MAPK inhibitor SB203580 selectively blocked Mural Cell recruitment, resulting in the formation of naked endothelial tubes without Mural Cells. SB203580 inhibited angiopoietin-1-induced Mural Cell recruitment without influencing angiopoietin-1-stimulated endothelial sprouting. Adenoviral vector-mediated expression of a dominant negative form of p38 MAPK significantly reduced Mural Cell recruitment, whereas overexpression of a constitutively activated form of MKK6, an upstream activator of p38 MAPK, increased Mural Cell number. These results indicate that the p38 MAPK signaling pathway plays a critical role in Mural Cell recruitment during neovascularization and may represent a therapeutic target in angiogenesis-related disorders.
