Recruitment Of Smooth Muscle Cells Research Articles

Abstract 20373: Inhibition of Glycogen Synthase Kinase-3β Amplifies β-catenin Signaling to Promote Dedifferentiation of Endothelial Cells to Improve Neovessel Formation

Rationale: Wnt signaling plays a key role in the development and the regeneration of neovessels, however, the underlying mechanisms of these processes are not clearly understood. Goal: To elucidate the mechanism of dedifferentiation of venous endothelial cells (VECs) to arterial (A)ECs. Methods and Results: Herein we demonstrate that 6-bromoindirubin-3'-oxime (BIO), an inhibitor of Glycogen Synthase Kinase (GSK)-3β elicits the stabilization and the nuclear accumulation of β-catenin, thereby its direct interaction with the transcription factor NANOG in the nucleus of ECs. In a ChiP experiment, NANOG protein bound to the NANOG-, BRACHYURY-, CD133- and VEGFR2 -promoters in these cells. In a cell-based assay, BIO induced the activation of the NANOG -promoter-luciferase reporter system. Accordingly, stimulation of VECs with BIO increased Notch-1, Ephrin-B2 and Hey2 expressions, indicating dedifferentiation these cells to AECs. While NANOG-depletion decreased BIO-induced NOTCH-1 expression and neovascularization. BIO increased Matrigel plug neovessel formation in nude mice. Immunostaining of Matrigel plug sections prepared from these mice showed increased expression of Notch-1, Ephrin-B2 and Hey2, indicating dedifferentiation of a subset of VECs into AECs. In a mouse model of hind limb ischemia, BIO induced the incorporation of VECs into collateral neovessels as characterized by Ephrin-B2- and Hey2-positivities, and smooth muscle cell recruitment. Summary: Thus, we show that a subset of mature VECs may retain the ability to dedifferentiate into AECs in vivo and suggest critical roles of GSK-3β inhibition in this process.

Tbx5 Is Required for Avian and Mammalian Epicardial Formation and Coronary Vasculogenesis

Holt-Oram syndrome is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. Overexpression of Tbx5 in the chick proepicardial organ impaired coronary blood vessel formation. However, the potential activity of Tbx5 in the epicardium itself, and the role of Tbx5 in mammalian coronary vasculogenesis, remains largely unknown. To evaluate the consequences of altered Tbx5 gene dosage during proepicardial organ and epicardial development in the embryonic chick and mouse. Retroviral-mediated knockdown or upregulation of Tbx5 expression in the embryonic chick proepicardial organ and proepicardial-specific deletion of Tbx5 in the embryonic mouse (Tbx5(epi-/)) impaired normal proepicardial organ cell development, inhibited epicardial and coronary blood vessel formation, and altered developmental gene expression. The generation of epicardial-derived cells and their migration into the myocardium were impaired between embryonic day (E) 13.5 to 15.5 in mutant hearts because of delayed epicardial attachment to the myocardium and subepicardial accumulation of epicardial-derived cells. This caused defective coronary vasculogenesis associated with impaired vascular smooth muscle cell recruitment and reduced invasion of cardiac fibroblasts and endothelial cells into myocardium. In contrast to wild-type hearts that exhibited an elaborate ventricular vascular network, Tbx5(epi-/-) hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by hypoxia inducible factor-1α upregulation and increased binding of hypoxyprobe-1. Tbx5(epi-/-) mice with such myocardial hypoxia exhibited reduced exercise capacity when compared with wild-type mice. Our findings support a conserved Tbx5 dose-dependent requirement for both proepicardial and epicardial progenitor cell development in chick and in mouse coronary vascular formation.

Sonic hedgehog mediates a novel pathway of PDGF-BB–dependent vessel maturation

AbstractRecruitment of mural cells (MCs), namely pericytes and smooth muscle cells (SMCs), is essential to improve the maturation of newly formed vessels. Sonic hedgehog (Shh) has been suggested to promote the formation of larger and more muscularized vessels, but the underlying mechanisms of this process have not yet been elucidated. We first identified Shh as a target of platelet-derived growth factor BB (PDGF-BB) and found that SMCs respond to Shh by upregulating extracellular signal-regulated kinase 1/2 and Akt phosphorylation. We next showed that PDGF-BB–induced SMC migration was reduced after inhibition of Shh or its signaling pathway. Moreover, we found that PDGF-BB–induced SMC migration involves Shh-mediated motility. In vivo, in the mouse model of corneal angiogenesis, Shh is expressed by MCs of newly formed blood vessels. PDGF-BB inhibition reduced Shh expression, demonstrating that Shh is a target of PDGF-BB, confirming in vitro experiments. Finally, we found that in vivo inhibition of either PDGF-BB or Shh signaling reduces NG2+ MC recruitment into neovessels and subsequently reduces neovessel life span. Our findings demonstrate, for the first time, that Shh is involved in PDGF-BB–induced SMC migration and recruitment of MCs into neovessels and elucidate the molecular signaling pathway involved in this process.

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

Trkb Signaling in Pericytes Is Required for Cardiac Microvessel Stabilization

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb −/−) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb−/− embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.

Absence of Chemokine (C-X-C Motif) Ligand 10 Diminishes Perfusion Recovery After Local Arterial Occlusion in Mice

In arteriogenesis, pre-existing anastomoses undergo enlargement to restore blood flow in ischemic tissues. Chemokine (C-X-C motif) ligand 10 (CXCL10) is secreted after Toll-like receptor activation. Toll-like receptors are involved in arteriogenesis; however, the role of CXCL10 is still unclear. In this study, we investigated the role for CXCL10 in a murine hindlimb ischemia model. Unilateral femoral artery ligation was performed in wild-type (WT) and CXCL10(-/-) knockout (KO) mice and perfusion recovery was measured using laser-Doppler perfusion analysis. Perfusion recovery was significantly lower in KO mice compared with WT at days 4 and 7 after surgery (KO versus WT: 28±5% versus 81±13% at day 4; P=0.003 and 57±12% versus 107±8% at day 7; P=0.003). Vessel measurements of α-smooth muscle actin-positive vessels revealed increasing numbers in time after surgery, which was significantly higher in WT when compared with that in KO. Furthermore, α-smooth muscle actin-positive vessels were significantly larger in WT when compared with those in KO at day 7 (wall thickness, P<0.001; lumen area, P=0.003). Local inflammation was assessed in hindlimb muscles, but this did not differ between WT and KO. Chimerization experiments analyzing perfusion recovery and histology revealed an equal contribution for bone marrow-derived and circulating CXCL10. Migration assays showed a stimulating role for both intrinsic and extrinsic CXCL10 in vascular smooth muscle cell migration. CXCL10 plays a causal role in arteriogenesis. Bone marrow-derived CXCL10 and tissue-derived CXCL10 play a critical role in accelerating perfusion recovery after arterial occlusion in mice probably by promoting vascular smooth muscle cell recruitment and maturation of pre-existing anastomoses.

Statins attenuate the development of atherosclerosis and endothelial dysfunction induced by exposure to urban particulate matter (PM10)

Exposure to ambient air particulate matter (particles less than 10μm or PM10) has been shown to be an independent risk factor for the development and progression of atherosclerosis. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have well-established anti-inflammatory properties. The aim of this study was to determine the impact of statins on the adverse functional and morphological changes in blood vessels induced by PM10. New Zealand White rabbits fed with a high fat diet were subjected to balloon injury to their abdominal aorta followed by PM10/saline exposure for 4weeks±lovastatin (5mg/kg/day) treatment. PM10 exposure accelerated balloon catheter induced plaque formation and increased intimal macrophages and lipid accumulation while lovastatin attenuated these changes and promoted smooth muscle cell recruitment into plaques. PM10 impaired vascular acetylcholine (Ach) responses and increased vasoconstriction induced by phenylephrine as assessed by wire myograph. Supplementation of nitric oxide improved the impaired Ach responses. PM10 increased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in blood vessels and increased the plasma levels of endothelin-1 (ET-1). Incubation with specific inhibitors for iNOS, COX-2 or ET-1 in the myograph chambers significantly improved the impaired vascular function. Lovastatin decreased the expression of these mediators in atherosclerotic lesions and improved endothelial dysfunction. However, lovastatin was unable to reduce blood lipid levels to the baseline level in rabbits exposed to PM10. Taken together, statins protect against PM10-induced cardiovascular disease by reducing atherosclerosis and improving endothelial function via their anti-inflammatory properties.

The role of oxidized low-density lipoproteins in atherosclerosis: the myths and the facts.

The oxidative modification hypothesis of atherosclerosis, which assigns to oxidized low-density lipoproteins (LDLs) a crucial role in atherosclerosis initiation and progression, is still debated. This review examines the role played by oxidized LDLs in atherogenesis taking into account data derived by studies based on molecular and clinical approaches. Experimental data carried out in cellular lines and animal models of atherosclerosis support the proatherogenic role of oxidized LDLs: (a) through chemotactic and proliferating actions on monocytes/macrophages, inciting their transformation into foam cells; (b) through stimulation of smooth muscle cells (SMCs) recruitment and proliferation in the tunica intima; (c) through eliciting endothelial cells, SMCs, and macrophages apoptosis with ensuing necrotic core development. Moreover, most of the experimental data on atherosclerosis-prone animals benefiting from antioxidant treatment points towards a link between oxidative stress and atherosclerosis. The evidence coming from cohort studies demonstrating an association between oxidized LDLs and cardiovascular events, notwithstanding some discrepancies, seems to point towards a role of oxidized LDLs in atherosclerotic plaque development and destabilization. Finally, the results of randomized clinical trials employing antioxidants completed up to date, despite demonstrating no benefits in healthy populations, suggest a benefit in high-risk patients. In conclusion, available data seem to validate the oxidative modification hypothesis of atherosclerosis, although additional proofs are still needed.

Abstract 18734: KLF4 Regulates Aortic Aneurysm Morphology and Deletion Attenuates Aortic Aneurysm Formation

Objective: Kruppel-like Factor 4 (KLF4) is known to play a role in inducing phenotypic modulation of smooth muscle cells (SMC) following ischemia reperfusion injury and in atherosclerosis; however, its role in aortic aneurysms (AA) remains unknown. The study objective was to evaluate the role of genetic deletion of KLF4 on development of experimental AAs. Methods: 8-12 week male (n=12) KLF4 flx/wt ERT tamoxifen Cre+ mice (“KLF4 conditional het KO mice”) and KLF4 flx/wt ERT tamoxifen Cre- (WT) mice underwent elastase perfusion following tamoxifen injection. In separate experiments, 8-12 week male (n=13) KLF4 flx/flx Smooth muscle myosin heavy chain (Myh11) Cre+(“SMC specific conditional KLF4 KO mice”), and KLF4 wt/wt SM-MHC Cre+ (“Myh11 Cre WT”) underwent elastase perfusion following tamoxifen administration. At 14 days, maximal aortic dilation was measured with video micrometry. Aortic samples were evaluated with histology for α-actin, macrophages, neutrophils, T lymphocytes and elastin. Results: Maximal aortic dilation was significantly reduced in KLF4 conditional het KO mice compared to WT controls (42.9 ± 7.3% versus 78.9 ± 12.6%, p&lt;0.039). In addition, these mice had reduced macrophage and neutrophil infiltration with greater elastin preservation and α-actin expression. SMC specific conditional KLF4 KO mice also showed a significant reduction in aortic dilation following elastase perfusion as compared to Myh11 Cre+ WT controls (53.4 ± 7.76% versus 93.8 ± 16.2%, p&lt;0.028). These mice also had reduced macrophage and neutrophil staining and increased elastin preservation and α-actin expression. Conclusions: KLF4 plays a critical role in promoting AA formation, since heterozygous KLF4 KO mice showed reduced aneurysm formation. At least part of this effect is due to loss of KLF4 in SMC since SMC specific conditional KLF4 KO mice also showed reduced AA formation as well as decreased immune cell recruitment and greater preservation of elastin and SMCs. Taken together, results identify KLF4 as a novel therapeutic target for treatment of aneurysms.

Role of LRP1 in Protecting the Vasculature

Since the early studies of Russell Ross, numerous investigators have confirmed that inflammation is a driving force behind chronic diseases such as atherosclerosis, and key to this process is the recruitment and activation of macrophages. Our studies reveal that the LDL receptor-related protein (LRP1) plays an important role in macrophage biology by regulating the adhesive properties of the integrin α M ß 2 . LRP1 is a large endocytic and signaling receptor that is widely expressed and mediates the endocytosis and subsequent degradation of several ligands. Deletion of the Lrp1 gene is lethal in mice, and genome wide association studies reveal that the Lrp1 gene represents a susceptibility locus for migraine, for elevated plasma lipids, and for coronary heart disease. LRP1 is abundantly expressed in macrophages where it functions to protect the vasculature from the development of disease. By employing macrophage specific knockout mice (macLRP1-/- mice) we discovered that LRP1 expressed in these cells protects the vasculature from excessive remodeling upon injury by regulating the TGFß signaling pathway and by influencing vascular smooth muscle cell (SMC) recruitment into the developing neo-intima. It is well established that an important contribution to cardiovascular diseases is the transdifferentiation of SMC. Under normal physiological conditions, SMC are fully differentiated and in a contractile phenotype. However, under pathological conditions SMC switch to a proliferative and migratory phenotype and contribute to lesion development in atherosclerosis and/or neo-intima formation. Significant effort is underway to define molecular mechanisms regulating SMC transdifferentiation, and substantial evidence suggests an important role for the LDL receptor related protein 1 (LRP1) in this process by modulating the PDGF signaling pathway. Our data in SMC LRP1 deficient mice (smLRP1-/-) reveal that LRP1 modulates SMC transdifferentiation and regulates pathways relevant to neo-intima and aneurysm formation.

STAT1 activation by venous malformations mutant Tie2-R849W antagonizes VEGF-A-mediated angiogenic response partly via reduced bFGF production

A missense mutation from arginine to tryptophan at residue 849 in the kinase domain of Tie2 (Tie2-R849W) is commonly identified in familial venous malformations. The mechanistic action of Tie2-R849W variant expression on angiogenic cascades including smooth muscle cell recruitment, however, remains elusive. To avoid confounding factors from endogenous Tie2 expression, Tie2-depleted endothelial cells (ECs) were used to study the effects of ectopic shRNA-resistant Tie2 variant expression, Tie2-WT* and Tie2-R849W*, on vascular cell proliferation, migration, tube formation, and smooth muscle cell (SMC) recruitment. Tie2-R849W* induced STAT1 phosphorylation at Tyr701. Tie2-R849W*-expressing cells had reduced ability to migrate and form tubes on Matrigel than their wildtype counterparts. STAT1 phosphorylation attenuated VEGF-A-induced STAT3 tyrosine phosphorylation in Tie2-R849W*-expressing HUVECs. The induced STAT1 activation also decreased VEGF-A-induced bFGF mRNA expression by competing with activated STAT3 for a direct binding to the consensus STAT-binding site at positions -997 to -989 bp from transcription start site in the bFGF promoter. Depleting STAT1 expression rescued the inability of Tie2-R849W expression to mediate angiogenesis. Moreover, bFGF neutralization or constitutive STAT1 activation, reminiscence of Tie2-R849W* expression, suppressed the smooth muscle cell recruiting ability of endothelial conditioned medium. This work reveals an anti-angiogenic role of STAT1 activation that acts in Tie2-R849W-expressing ECs to impair VEGF-A-mediated STAT3 signaling, bFGF production, and smooth muscle cell recruitment. A balancing activity of STAT1 and STAT3 may be important for Tie2-mediated vascular homeostasis.

Novel Role of Proline-Rich Nonreceptor Tyrosine Kinase 2 in Vascular Wall Remodeling After Balloon Injury

To investigate the role of Pyk2, a proline-rich nonreceptor tyrosine kinase, in G protein-coupled receptor agonist, thrombin-induced human aortic smooth muscle cell growth and migration, and injury-induced vascular wall remodeling. Thrombin, a G protein-coupled receptor agonist, activated Pyk2 in a time-dependent manner and inhibition of its stimulation attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. Thrombin also activated Grb2-associated binder protein 1, p115 Rho guanine nucleotide exchange factor, Rac1, RhoA, and p21-activated kinase 1 (Pak1) and interference with stimulation of these molecules attenuated thrombin-induced human aortic smooth muscle cell migration and proliferation. In addition, adenovirus-mediated expression of dominant negative Pyk2 inhibited thrombin-induced Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA and Pak1 stimulation. Balloon injury also caused activation of Pyk2, Grb2-associated binder protein 1, p115 rho guanine nucleotide exchange factor, Rac1, RhoA, and Pak1 in the carotid artery of rat, and these responses were sensitive to inhibition by the dominant negative Pyk2. Furthermore, inhibition of Pyk2 activation resulted in reduced recruitment of smooth muscle cells onto the luminal surface and their proliferation in the intimal region leading to suppression of neointima formation. Together, these results demonstrate for the first time that Pyk2 plays a crucial role in G protein-coupled receptor agonist thrombin-induced human aortic smooth muscle cell growth and migration, as well as balloon injury-induced neointima formation.

Smooth muscle–endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.

The double‐edged sword of fibronectin in atherosclerosis

Atherosclerosis, the leading cause of death in Westernized countries, derives from a non‐resolving inflammation of the large arteries. A critical step in the initiation of an atherosclerotic plaque is the expansion of intimal space (the area between the endothelial cell (EC) lining and the medial smooth muscle cell layer) in susceptible regions of arteries (Tabas et al, 2007). The disease‐prone locations are largely determined by arterial blood flow characteristics, with branch points and the lesser curvatures of arteries, where the flow is turbulent, being particularly disposed to plaque formation. At these sites, the normally thin intimal space expands with the deposition of extracellular matrix (ECM), which then serves as a sticky surface to trap LDL particles that are constantly crossing the endothelial layer to enter the artery. One reason for this retention is that the apoB100 molecule on the LDL particle has a specific domain that binds tightly to the ECM, and genetic causation studies in a mouse model of atherosclerosis have shown that when this domain is deleted, plaque burden is significantly reduced despite equivalent hyperlipidemia (Skalen et al, 2002). As implied by the ‘Response to Retention’ hypothesis (Tabas et al, 2007), the retained lipoprotein particles set in motion the invasion of the arterial wall by monocytes that quickly become macrophages that engorge themselves on cholesterol and the other lipids to form foam cells. Foam cells not only physically expand the plaque, but by secreting a variety of chemokines and cytokines, they also create and amplify an inflammatory state (Fig 1). Figure 1. Plasma derived FN promotes atherogenesis in mouse models of atherosclerosis. pFN, synthesized in the liver, is deposited in susceptible regions of the arteries and contributes to early atherosclerotic lesion formation and the recruitment of smooth muscle cells that shape the fibrous cap of advanced lesions (upper panel). …

Trapped in the Plaque

Netrin-1 enhances the retention of macrophages and the recruitment of smooth muscle cells, thereby promoting the progression of atherosclerotic lesions.

Tumor vessel stabilization and remodeling by anti-angiogenic therapy with bevacizumab

Bevacizumab-resistant tumor vessels were characterized by an increased vessel diameter and normalization of vascular structures by the recruitment of mature pericytes and smooth muscle cells. Here, we analyzed human liver metastases which were taken at clinical relapse in patients with colorectal adenocarcinoma treated with anti-angiogenic therapy using the humanized monoclonal anti-VEGF bevacizumab. Tumor vessels which are resistant to anti-VEGF therapy are increased in size and characterized by a normalization of the vascular bed. These results were confirmed using NOD SCID mice as animal model and xenograft transplantation of human PC-3 prostate carcinoma cells in combination with bevacizumab treatment. Our results confirmed that anti-angiogenic therapy results in enhanced vascular remodeling by vascular stabilization. This process is apparently accompanied by enhanced necrosis of tumor tissue. These processes interfere with the efficacy of anti-angiogenic therapy because of reduced susceptibility of stabilized vessels by this therapy. These results demonstrate the importance for the development of second generation anti-angiogenic combination therapy concepts to rule out the balance between vascular stabilization followed by a possible de-stabilization making the remained vessels susceptible to a second wave of anti-angiogenic therapy.

Blood Coagulation and Blood Vessel Development

In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Arderiu et al1 investigate the role of tissue factor (TF) in angiogenesis using both in vitro and in vivo models. They find that TF expression in endothelial cells (ECs) stimulates the expression of chemokine ligand 2 (CCL2). This facilitates the recruitment of vascular smooth muscle cells (VSMCs) and the stabilization of EC-VSMC networks. See accompanying article on page 2607 Judah Folkman, a pioneer in angiogenesis research, was intrigued by the connection between blood coagulation and blood vessel development and proposed that the 2 processes were intimately connected.2,3 A summary of the key observations connecting TF and angiogenesis is shown in the Figure. In 1994, Zhang and colleagues4 were the first to show that TF expression by Meth-A sarcoma cells regulates their angiogenic activity in vivo. A high level of TF expression was associated with enhanced expression of the proangiogenic factor vascular EC growth factor. Later, Yu et al5 demonstrated that antisense silencing of TF expression in a human colorectal cancer cell line reduced the growth of tumors in mice. A study with human MDA-MB-231 breast cancer cells revealed that the TF/factor VIIa complex regulated the expression of interleukin-8, another angiogenic factor, via activation of protease-activated receptor 2 (PAR2).6 Interestingly, the transcriptional program induced by activation of PAR2 was similar to that induced by activation of the thrombin receptor PAR1 and included many angiogenic factors and chemokines.7 A summary of the proposed coagulation protease-PAR pathways that lead to the expression of angiogenic factors by tumors cells is shown in panel A of the Figure. One area of controversy is whether TF is expressed by ECs within tumors. One study8 reported TF expression by ECs in invasive breast cancer but not …

Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2(+/-)) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2(+/-) mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2(+/-) macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

Angiopoietin-1 and -2 Exert Antagonistic Functions in Tumor Angiogenesis, yet Both Induce Lymphangiogenesis

Members of the Angiopoietin family regulate various aspects of physiologic and pathologic angiogenesis. Although Angiopoietin-1 (Ang-1) decreases endothelial cell permeability and increases vascular stabilization via recruitment of pericytes and smooth muscle cells to growing blood vessels, Angiopoietin-2 (Ang-2) mediates angiogenic sprouting and vascular regression. In this study, we used the Rip1Tag2 transgenic mouse model of pancreatic β-cell carcinogenesis to investigate the roles of Ang-1 and Ang-2 in tumor angiogenesis and tumor progression. On their own, transgenic expression of human Ang-1 or Ang-2 in pancreatic β cells caused formation of peri-insular lymphatic vessels in the absence of effects on blood vessel density, islet morphology, or physiology. When crossed to Rip1Tag2 mice, both Ang-1-and Ang-2-expressing β-cell tumors showed increased peritumoral lymphangiogenesis in the absence of metastasis to local lymph nodes or distant organs. There was no alteration in tumor outgrowth, blood vessel density, or vessel maturation in Ang-1-expressing tumors. In contrast, Ang-2-expressing tumors exhibited diminished pericyte recruitment to blood vessels that were dilated, nonfunctional, and highly permeable. These tumors were hemorrhagic, highly infiltrated by leukocytes, and impaired in outgrowth. Together, our findings establish that Ang-2 antagonizes Ang-1 function, leading to excessive vessel sprouting with impaired pericyte recruitment and vessel stabilization. The poor perfusion of immature blood vessels results in retarded tumor growth, defining an important pathophysiologic pathway required for efficient tumorigenesis.

Focal Adhesion Kinase Regulates Smooth Muscle Cell Recruitment to the Developing Vasculature

The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. We crossed fak(loxp) targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAK(wnt) and FAK(nk)) or coronary SMC (FAK(cSMC)). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with postnatal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, platelet-derived growth factor PDGFBB. FAK depletion resulted in unstable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1, and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in stimulated extracellular matrix degradation. FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus.