Hideto Sano

Cyclic AMP Potentiates Vascular Endothelial Cadherin-Mediated Cell-Cell Contact To Enhance Endothelial Barrier Function through an Epac-Rap1 Signaling Pathway

ABSTRACT Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′, 5′-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.

Endothelial HIF-2 mediates protection and recovery from ischemic kidney injury

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitor-treated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

The Foxc2 Transcription Factor Regulates Angiogenesis via Induction of Integrin β3 Expression

Forkhead transcription factor Foxc2 is an essential regulator of the cardiovascular system in development and disease. However, the cellular and molecular functions of Foxc2 in vascular endothelial cells are still not fully understood. Here, through gene expression profiling in endothelial cells, we identified molecules associated with cell-extracellular matrix interactions, integrin β3 (Itgb3), integrin β5 (Itgb5), and fibronectin, as downstream targets of Foxc2. We found that Itgb3 expression is directly regulated by Foxc2 through multiple Forkhead-binding elements within two high homology regions in the Itgb3 promoter. Because Itgb3 is known to regulate angiogenesis, we further tested whether Foxc2 is directly involved in angiogenesis by regulating Itgb3 expression by in vitro experiments. Overexpression of Foxc2 significantly enhanced endothelial cell migration and adhesion, whereas this effect was strongly inhibited by Itgb3 neutralization antibody. In accordance with these results, pulmonary microvascular endothelial cells isolated from Foxc2 heterozygous mutant mice showed a marked reduction in Itgb3 expression and cell migration. Finally, ex vivo aortic ring assay to test the sprouting and microvessel formation revealed enhanced microvessel outgrowth by Foxc2 overexpression. Conversely, microvessel outgrowth from aortas of Foxc2 heterozygous mutant mice was reduced. Taken together, these results suggest that Foxc2 directly induces Itgb3 expression and regulates angiogenesis by Itgb3-mediated endothelial cell adhesion and migration.

The Foxc2 transcription factor regulates tumor angiogenesis

The Forkhead/Fox transcription factor Foxc2 is a critical regulator of vascular development. However, the role of Foxc2 in pathological angiogenesis in cancer remains unknown. Here we show that FoxC2 is highly expressed in human breast and colonic tumors and in the tumor endothelium in human and mouse melanomas. Using the B16 melanoma tumor model, we investigated the function of Foxc2 in tumor angiogenesis. After subcutaneous injection of B16 melanoma cells, primary tumor growth as well as neovascularization was markedly reduced in mice lacking one copy of the Foxc2 gene (Foxc2+/-). Consistently, expression levels of several angiogenic factors, including vascular endothelial growth factor (Vegf), matrix metallopeptidase 2 (Mmp2), and platelet-derived growth factor-B (Pdgfb), were significantly decreased in B16 tumors grown in Foxc2+/- mice, and tumor blood vessels formed in Foxc2+/- mice showed reduced coverage of mural cells and endothelial cell apoptosis. In addition, the tumor tissue in Foxc2+/- mice had an accumulation of necrotic cells. Taken together, these findings demonstrate that haplodeficiency of Foxc2 results in impaired formation of tumor blood vessels as well as reduced tumor growth and thereby provide evidence that Foxc2 is critical for tumor development and angiogenesis.

Role of Bone Marrow-Derived Progenitor Cells in Cuff-Induced Vascular Injury in Mice

Objectives—Arterial injury results in vascular remodeling associated with proliferation and migration of smooth muscle cells (SMCs) and the development of intimal hyperplasia, which is a critical component of restenosis after angioplasty of human coronary arteries and an important feature of atherosclerotic lesions. However, the origin of SMCs and other cells in the development of vascular remodeling is not yet fully understood. Methods and Results—We utilized a cuff-induced vascular injury model after transplantation of the bone marrow (BM) from green fluorescent protein (GFP)-transgenic mice. We found that macrophages were major cells recruited to the adventitia of the vascular injury lesion along with SMCs and endothelial cells (ECs). While investigating whether those cells are derived from the donor, we found that most of the macrophages were GFP-positive, and some of the SMCs and ECs were also GFP-positive. Administration of the anti–c-fms antibody resulted in a marked decrease in macrophages and a relative increase of SMCs, while administration of antibodies against the platelet-derived growth factor receptor-&bgr; caused a prominent decrease in SMCs and a relative increase in macrophages. Conclusions—The current study indicates that BM-derived cells play an important role in vascular injury, and that differentiation of macrophages and SMCs might be dependent on each other.

Negative Regulation of VEGF-Induced Vascular Leakage by Blockade of Angiotensin II Type 1 Receptor

Objective—Permeability of blood vessels is essential for tissue homeostasis. However, disorganized hyperpermeability leads to progression of diseases. Vascular endothelial growth factor-A (VEGF) is a key regulator for leakiness of blood vessels and it has been reported that VEGF-mediated hyperpermeability was suppressed by angiopoietin-1 (Ang1). We found that Angiotensin-converting enzyme (ACE) was downregulated in endothelial cells by Ang1. ACE converts angiotensin I to angiotensin II (AII). Here, we studied the relationship between VEGF and AII relative to vascular permeability. Methods and Results—We showed that VEGF-mediated vascular hyperpermeability was suppressed in mice given AII type 1 receptor (AT1R) blocker (ARB); the effect was also seen in AT1R-deficient mice. In this system, we found that ARB inhibited VEGF-induced gap formation. Furthermore, we ascertained that angioedema induced by overexpression of VEGF decreased noticeably in ARB-treated ischemic mice. Conclusions—Because ARB suppressed VEGF-induced vascular hyperpermeability, we propose that ARB may be used to minimize the risk of edema in therapeutic angiogenesis using VEGF.

Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an αIIbβ3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Endogenously Generated Plasmin at the Vascular Wall Injury Site Amplifies Lysine Binding Site-Dependent Plasminogen Accumulation in Microthrombi

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

TM5275 prolongs secreted tissue plasminogen activator retention and enhances fibrinolysis on vascular endothelial cells

INTRODUCTION Elevated plasminogen activator inhibitor-1 (PAI-1) reduces fibrinolytic potential in plasma, contributing to thrombotic disease. Thus, inhibiting PAI-1 activity is clinically desirable. We recently demonstrated that tissue plasminogen activator (tPA) remains on the surface of vascular endothelial cells (VECs) after secretion in a heavy-chain dependent manner, which is essential for high fibrinolytic activity on the surface of VECs, and that PAI-1 dissociates retained tPA from the cell surface as a result of high-molecular weight complex formation. Based on the model whereby amounts of tPA and its equilibrium with PAI-1 dynamically change after exocytosis, we examined how TM5275, a newly synthesized small molecule PAI-1 inhibitor, modulated tPA retention and VEC surface-derived fibrinolytic activity using microscopic techniques. MATERIALS AND METHODS The effects of TM5275 on the kinetics of the secretion and retention of green fluorescent protein (GFP)-tagged tPA (tPA-GFP) on VECs were analyzed using total internal reflection fluorescence microscopy. The effects of TM5275 on the generation of plasmin activity were evaluated by both plasminogen accumulation and fibrin clot lysis on tPA-GFP-expressing VECs using confocal laser scanning microscopy. RESULTS TM5275 at concentrations of 20 and 100 μM significantly prolonged the retention of tPA-GFP on VECs by inhibiting tPA-GFP-PAI-1 high-molecular-weight complex formation. TM5275 enhanced the time-dependent accumulation of plasminogen as well as the dissolution of fibrin clots on and around the tPA-GFP-expressing cells. CONCLUSIONS The profibrinolytic effects of TM5275 were clearly demonstrated by the prolongation of tPA retention and enhancement of plasmin generation on the VEC surface as a result of PAI-1 inhibition.

Lysophosphatidylcholine Acyltransferase-3 Expression Is Associated with Atherosclerosis Progression

Free arachidonic acid (AA) is an important precursor of lipid mediators such as leukotrienes and prostaglandins that induces inflammation and is associated with atherosclerosis progression. Recent studies have shown that lysophosphatidylcholine acyltransferase-3 (LPCAT3) converts lysophosphatidylcholine (LPC) and free AA into phosphatidylcholine (PC)-containing AA (arachidonyl-PC) and thereby can regulate intracellular free-AA levels. However, the association between LPCAT3 and atherosclerosis remains to be established. In this study, we analyzed human and mouse atherosclerotic tissues to gain insight into the arachidonyl-PC metabolism involving LPCAT3 using imaging mass spectrometry. The data revealed a complementary distribution of arachidonyl-PC and LPC in human atherosclerotic tissues with arachidonyl-PC decreasing and LPC increasing as atherosclerosis progressed. Furthermore, we found a homologous distribution of LPCAT3 expression and arachidonyl-PC based on atherosclerotic progression. In contrast, in ApoE-deficient mice, atherosclerosis increased both arachidonyl-PC accumulation and LPCAT3 expression. Taken together, these findings suggest that the regulation of LPCAT3 expression might be associated with atherosclerotic progression in humans.