Induced Endothelial Cells Migration Research Articles

Oroxylin a Attenuates Limb Ischemia by Promoting Angiogenesis via Modulation of Endothelial Cell Migration.

Oroxylin A (OA) has been shown to simultaneously increase coronary flow and provide a strong anti-inflammatory effect. In this study, we described the angiogenic properties of OA. OA treatment accelerated perfusion recovery, reduced tissue injury, and promoted angiogenesis after hindlimb ischemia (HLI). In addition, OA regulated the secretion of multiple cytokines, including vascular endothelial growth factor A (VEGFA), angiopoietin-2 (ANG-2), fibroblast growth factor-basic (FGF-2), and platelet derived growth factor BB (PDGF-BB). Specifically, those multiple cytokines were involved in cell migration, cell population proliferation, and angiogenesis. These effects were observed at 3, 7, and 14 days after HLI. In skeletal muscle cells, OA promoted the release of VEGFA and ANG-2. After OA treatment, the conditioned medium derived from skeletal muscle cells was found to significantly induce endothelial cell (EC) proliferation. OA also induced EC migration by activating the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinase 2 (ROCK-II) signaling pathway and the T-box20 (TBX20)/prokineticin 2 (PROK2) signaling pathway. In addition, OA was able to downregulate the number of macrophages and neutrophils, along with the secretion of interleukin-1β, at 3 days after HLI. These results expanded current knowledge about the beneficial effects of OA in angiogenesis and blood flow recovery. This research could open new directions for the development of novel therapeutic intervention for patients with peripheral artery disease (PAD).

Endothelial specific YY1 deletion restricts tumor angiogenesis and tumor growth

Angiogenesis is a physiological process for the formation of new blood vessels from the pre-existing vessels and it has a vital role in the survival and growth of neoplasms. During tumor angiogenesis, the activation of the gene transcriptions in vascular endothelial cells (ECs) plays an essential role in the promotion of EC proliferation, migration, and vascular network development. However, the molecular mechanisms underlying transcriptional regulation of EC and tumor angiogenesis remains to be fully elucidated. Here we report that the transcription factor Yin Yang 1 (YY1) in ECs is critically involved in tumor angiogenesis. First, we utilized a tamoxifen-inducible EC-specific YY1 deficient mouse model and showed that YY1 deletion in ECs inhibited the tumor growth and tumor angiogenesis. Using the in vivo matrigel plug assay, we then found that EC-specific YY1 ablation inhibited growth factor-induced angiogenesis. Furthermore, vascular endothelial growth factor (VEGF)-induced EC migration was diminished in YY1-depleted human umbilical vein endothelial cells (HUVECs). Finally, a rescue experiment revealed that YY1-regulated BMP6 expression in ECs was involved in EC migration. Collectively, our results demonstrate that endothelial YY1 has a crucial role in tumor angiogenesis and suggest that targeting endothelial YY1 could be a potential therapeutic strategy for cancer treatment.

Selective increase of cardiomyocyte derived extracellular vesicles after experimental myocardial infarction and functional effects on the endothelium

IntroductionWound healing after myocardial infarction (MI) is mediated by different cell types, secreted proteins, components of the extracellular matrix (ECM) and, as increasing evidences suggest, extracellular vesicles (EVs). We aim to determine the dynamics of release and origin of EVs after MI, as well as their biological activity on endothelial cells (ECs). MethodsMI was induced in WT mice and blood and tissues collected at baseline, 3, 15 and 30 days post-ligation for cardiac function (echocardiography) and histological evaluation. Circulating EVs subpopulations were measured by flow cytometry in mouse, and in a small cohort of patients with ST-segment elevation MI (STEMI, n = 6). In vitro, EVs were isolated from a cardiomyocyte cell line (HL1) and their function assayed on ECs. ResultsLeukocyte and endothelial EVs increased concomitant to inflammatory and angiogenic processes triggered by ischemia. More strikingly, cardiomyocyte EVs (connexin43+) were detected in STEMI patients and in murine MI, where a significant increase in their levels was reported at day 15 post-ischemia (p < 0.05 vs baseline). In vitro, HL1EVs induced ECs migration (p = 0.05) and proliferation (p < 0.05), but impaired tube formation. These apparent contradictory results could be partially explained by the upregulation of MMP3, and the apoptosis and senescence genes, p53 and p16, induced by HL1EVs on ECs (p < 0.05). ConclusionsMI induces the release of different EVs subpopulations, including those of cardiac origin, in a preclinical model of MI and STEMI patients. In vitro, cardiomyocyte derived EVs are able to modulate endothelial function, suggesting their active role in heart repair after ischemia.

Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration

Background: Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Methods: Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. Results: S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P₁ receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P₂ or S1P₃ receptor had no effect. Conclusion: Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies.

Differential role of integrin beta 4 in HLA class-I induced endothelial cell activation

Aim The development of anti-donor HLA class I (HLA-I) antibodies following solid organ transplant is associated with acute and chronic rejection and graft failure. Following ligation with antibody, HLA class I molecules form a molecular partnership with integrin β 4 (ITGB4) to transduce activation and proliferative signals in endothelial cells (EC) contributing to rejection. We hypothesize that determination of the domains on the integrin β 4 molecule that interact with HLA-I will provide critical information for the development of novel therapeutics for the treatment of chronic rejection. Methods To identify the ITGB4 intracellular domain(s) that associates with HLA-I, we used the ITGB4 full length (Wild Type) construct or deletion mutants that lack the Calx- β , fibronectin type III repeat FNIII 1-4 or connecting segment (CS) domain . These constructs were sub-cloned into an adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. Primary human aortic endothelial cells (HAEC) lacking endogenous ITGB4 were infected with adenovirus and then EC activation was determined by analyzing protein phosphorylation, stress fiber formation, cell migration and proliferation. Results Our results demonstrate that HLA-I binds to ITGB4 via the CS domain. The CS domain is required for the phosphorylation of Src, Paxillin, Akt, ERK1/2 and S6RP. The FNIII 3&4 domains are required for phosphorylating Paxillin, Akt, ERK1/2 and S6RP, but not Src. HLA-I phosphorylates ITGB4 at Tyr1494 and Tyr1526 in a Src dependent manner. Furthermore, deletion of CS, FNIII3&4, Calx- β inhibited HLA-I induced EC stress fiber formation, migration and proliferation. Moreover, HLA-I induces association between ITGB4 and the adaptor protein Shc which depends on CS and FNIII3&4 domains. Suppression of endogenous Shc protein by siRNA inhibited HLA-I induced EC migration, proliferation and phosphorylation of Akt, ERK1/2 and S6RP, but not Src. Conclusion Understanding the molecular mechanisms underlying HLA antibody-induced EC proliferation and migration should enable the development of novel therapeutic strategies for the treatment of antibody-mediated chronic transplant rejection.

OR23: THE INTEGRIN β4 CONNECTING SEGMENT DOMAIN IS REQUIRED FOR HLA CLASS I-MEDIATED ENDOTHELIAL CELL ACTIVATION

Endothelial cell (EC) activation is a major consequence of HLA class I (HLA-I) antibody-mediated rejection. Previous studies have shown that the molecular association between HLA-I molecule and integrin β 4 (ITGB4) is required for EC migration and proliferation by regulating ERK1/2 signaling pathway. However, the proximal signaling pathway that regulates HLA-I and ITGB4 association remains unclear. We determined the domains of the ITGB4 cytoplasmic tail required for HLA-I mediated phosphorylation pathways and correlated these findings with functional studies. Primary human aortic endothelial cells (HAEC) were used for this study. To identify the ITGB4 intracellular domain that associates with HLA-I, we generated constructs encoding ITGB4 full length (WT) or deletion mutants that lack the Calx- β , fibronectin type III repeat (FNIII 1–4) or connecting segment (CS). These constructs were sub-cloned into adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. HAEC lacking endogenous ITGB4 were infected with adenovirus and protein phosphorylation and cell migration were further analyzed. Flow cytometry and Western blot analysis showed that both the WT and deletion mutant-infected HAEC had similar levels of cell surface or total expression of ITGB4. Coimmunoprecipitation studies showed that stimulation with the F(ab′)2 fragment of HLA-I antibody increased HLA-I association with ITGB4 in a time dependent manner in WT adenovirus infected HAEC. In contrast, HAEC infected with the CS deletion mutant significantly inhibited HLA-I association with ITGB4 compared to WT, Calx- β or FNIII mutant infected cells. In addition, the CS deletion mutant decreased HLA-I induced ERK1/2 phosphorylation as measured by Western blot. Using in vitro wound healing assay, we found that over expression of ITGB4 increased EC migration compared to LacZ infected cells, whereas CS deletion decreased HLA-I induced EC migration. Furthermore, HLA-I antibody increased the association of ITGB4 with the adaptor protein Shc. This association was decreased by CS deletion. Our results demonstrate that HLA-I binds to ITGB4 via the connecting segment domain that is required for ERK1/2 signaling and EC activation.

Endotoxin-induced vascular endothelial cell migration is dependent on TLR4/NF-κB pathway, NAD(P)H oxidase activation, and transient receptor potential melastatin 7 calcium channel activity

Endothelial dysfunction is decisive and leads to the development of several inflammatory diseases. Endotoxemia-derived sepsis syndrome exhibits a broad inflammation-induced endothelial dysfunction. We reported previously that the endotoxin, lipopolysaccharide (LPS), induces the conversion of endothelial cells (ECs) into activated fibroblasts, showing a myofibroblast-like protein expression profile. Enhanced migration is a hallmark of myofibroblast function. However, the mechanism involved in LPS-induced EC migration is no totally understood. Some studies have shown that the transient receptor potential melastatin 7 (TRPM7) ion channel is involved in fibroblast and tumor cell migration through the regulation of calcium influx. Furthermore, LPS modulates TRPM7 expression. However, whether TRPM7 is involved in LPS-induced EC migration remains unknown.Here, we study the participation of LPS as an inducer of EC migration and study the mechanism underlying evaluating the participation of the TRPM7 ion channel.Our results demonstrate that LPS induced EC migration in a dose-dependent manner. Furthermore, this migratory process was mediated by the TLR-4/NF-κB pathway and the generation of ROS through the PKC-activated NAD(P)H oxidase. In addition, LPS increased the intracellular calcium level and the number of focal adhesion kinase (FAK)-positive focal adhesions in EC. Finally, we demonstrate that using TRPM7 blockers or suppressing TRPM7 expression through siRNA successfully inhibits the calcium influx and the LPS-induced EC migration.These results point out TRPM7 as a new target in the drug design for several inflammatory diseases that impair vascular endothelium function.

Abstract 238: Angiogenesis Requires G-Protein-Coupled Receptor Kinase 2-Interacting Protein 1 (GIT1): A Novel GIT1-ERK1/2-Cortactin Pathway Controls Endothelial Cell Directional Migration

Introduction— GIT1 is a multidomain scaffold protein involved in diverse cellular processes including cell adhesion and migration. GIT1 is required for vascular endothelial cell growth factor (VEGF) signaling in endothelial cells (EC) and translocates to lamellipodia during directional migration. Lamellipodia are regions of very rapid actin remodeling which is mediated in part by the cortical actin remodeling protein, cortactin. We hypothesize that GIT1 mediates directional migration of EC by regulating cortactin localization and activation. Methods and Results— To determine the role of GIT1 in directional migration of EC, we used a Dunn’s chamber and in vitro scratch wound healing assay. In Dunn’s chamber, the migration of GIT1 siRNA transfected Human Umbilical Vein EC (HUVEC) was decreased by 66±9.1% compared to control siRNA transfected EC in response to 50ng/ml VEGF gradient. Furthermore, the extent of wound healing was decreased by 63±7.8% in GIT1 knock out (GIT1-KO) mouse lung microvascular EC (MLMEC). We next studied the localization pattern of cortactin in MLMEC from GIT1 wild type (GIT1-WT) and GIT1-KO mice. In GIT1-KO MLMEC the number of cells with cortactin localized by immunofluorescence (IF) at the leading edge during wound healing was reduced by 73±5.7% compared to GIT1-WT. Dual IF for cortactin and GIT1 at the leading edge of HUVEC showed extensive co-localization. In response to 50ng/ml VEGF for 15 min, serum deprived HUVEC exhibited a 75.6±11.3% increase in GIT1-cortactin association as demonstrated by co-immunoprecipitation (IP). Among GIT1 domains, the Spa homology domain (SHD) is unique to GIT1 and is required for signal transduction. We next transfected GIT1-KO MLMEC with GIT1(1-770), GIT1(1-420) that contains SHD, and GIT1(1-250+420-770) that lacks SHD. IF and IP analysis of MLMEC showed that SHD was required for cortactin membrane localization and cortactin-GIT1 association. Finally, we showed that GIT1 regulates VEGF induced ERK1/2 mediated phosphorylation of cortactin Ser405, a requirement for cortactin activation. Conclusion— The unique SHD of GIT1 is essential for VEGF induced EC migration by assembling a GIT1-ERK1/2-cortactin complex suggesting that SHD of GIT1 may be a therapeutic target to limit pathological angiogenesis.

Combination of IL-17 and TNFα induces a pro-inflammatory, pro-coagulant and pro-thrombotic phenotype in human endothelial cells

ObjectiveCardiovascular events remain the leading cause of death in rheumatoid arthritis (RA). To study the role of cytokines in these observations, the effects of tumour necrosis factor α (TNFα) and...

Hypoxia induces tumor and endothelial cell migration in a Semaphorin 3F- and VEGF-dependent manner via transcriptional repression of their common receptor Neuropilin 2

Neuropilin-2 (NRP2) is a receptor expressed by tumor cells and endothelial cells (EC) that binds both semaphorin 3F (SEMA3F), a potent inhibitor of tumor angiogenesis and metastasis, and vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. It was found that glioblastoma and melanoma cells repressed NRP2 expression when maintained under hypoxic conditions and after treatment with the hypoxia-mimetic agent desferrioxamine (DFO), at both the mRNA and protein levels. Silencing of HIF1-α, the hypoxia-induced subunit of the hypoxia inducible factor (HIF), abrogated DFO-induced NRP2 repression. Conversely, ectopic expression of HIF1-α directly repressed NRP2 promoter activity and expression. NRP2 is the sole receptor for SEMA3F. Loss of NRP2 expression in tumor cells inhibited SEMA3F-dependent activities, such as inactivation of RhoA, depolymerization of F-actin, and inhibition of tumor cell migration. On the other hand, loss of NRP2 expression in tumor cells increased VEGF protein levels in conditioned media, with no effects on VEGF mRNA levels. This increase in VEGF protein levels promoted paracrine activation of EC, including VEGF receptor-2 phosphorylation, and activation of downstream signaling proteins such as p44/42 MAPK and p38 MAPK. In addition, the elevated VEGF levels induced EC migration and sprouting, two key steps of tumor angiogenesis in vivo. It was concluded that hypoxia regulates VEGF and SEMA3F activities through transcriptional repression of their common receptor NRP2, providing a novel mechanism by which hypoxia induces tumor angiogenesis, growth and metastasis.

Abstract 3464: Melanomas differentially regulate directionally persistent endothelial cell migration

Abstract Introduction: Cutaneous melanoma is the most aggressive form of skin cancer. Although early detection and surgical resection of the primary lesion could improve survival, metastatic melanoma is by large refractory to therapy and predicts poor prognosis. Melanoma progression and metastatic dissemination relies on the process of angiogenesis. The angiogenic process is comprised of a stringent cascade of events ultimately resulting in a new vessel. Pathological (tumor) angiogenesis, however, is mediated by an array of angiogenic modulators produced by the tumor itself. Such tumor-associated modulators arbitrate the enhanced and awry proliferative, survival and migratory responses exhibited by endothelial cells, culminating in unregulated and unimpeded angiogenesis. Project Description: In the current study, we evaluated the angiogenic potential of human melanoma cell lines, based on their ability to regulate directionally persistent endothelial cell (EC) migration. Melanoma conditioned medium (CM) from three human melanoma cell lines (BLM, Mel57, 1F6), differing in their degree of aggressiveness and clinical staging, were used to assess the migratory responses of ECs in a 2-D migration assay, previously established by our group. This assay involves the use of the ‘ring and barrier’ system, wherein the ECs were cultured in a migration ring to generate a confluent area at the periphery and a cell free area in the center. EC migration in response to the tested CMs was monitored real-time, and various migration parameters were determined. Additionally, CM size fractions were tested to delineate the differences observed in migratory responses of the ECs to the tested CMs. Results and Conclusions: There was a clear correlation between EC migration and the degree of aggressiveness of the melanoma cell line used to generate the CM. While the CM of the highly aggressive BLM tumor cells induced a high migratory response in ECs, the CM of the less aggressive 1F6 cells had an inhibitory effect. The 1F6 CM was further size fractionated and it was observed that the factor/s responsible for this inhibition lay in the 10-30 kD range. Gene and protein expression verification helped identify a varied protein expression profile, which are secreted into the CM. Their expression inversely correlated with aggressiveness of the melanoma cell line and ability of the respective CMs to affect EC migration. CM of 1F6 cells silenced with shRNA induced EC migration, while both recombinant protein as well as the CM of BLM cells transfected with protein expressing plasmid mitigated EC migration. Taken together, these results demonstrate that the soluble protein in the CM inhibits the inherent propensity of endothelial cells to undergo directionally persistent migration and that this expression could impede EC melanoma-induced angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3464. doi:10.1158/1538-7445.AM2011-3464

Hypoxia Inducible Factor–Dependent Regulation of Angiogenesis by Nitro–Fatty Acids

Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 μmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.

VEGF and inhibitors of TGFβ type-I receptor kinase synergistically promote blood-vessel formation by inducing α5-integrin expression

Vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGFbeta) are potent regulators of angiogenesis. How VEGF and TGFbeta signaling pathways crosstalk is not well understood. Therefore, we analyzed the effects of the TGFbeta type-I-receptor inhibitors (SB-431542 and LY-2157299) and VEGF on endothelial cell (EC) function and angiogenesis. We show that SB-431542 dramatically enhances VEGF-induced formation of EC sheets from fetal mouse metatarsals. Sub-optimal doses of VEGF and SB-431542 synergistically induced EC migration and sprouting of EC spheroids, whereas overexpression of a constitutively active form of TGFbeta type-I receptor had opposite effects. Using quantitative PCR, we demonstrated that VEGF and SB-431542 synergistically upregulated the mRNA expression of genes involved in angiogenesis, including the integrins alpha5 and beta3. Specific downregulation of alpha5-integrin expression or functional blocking of alpha5 integrin with a specific neutralizing antibody inhibited the cooperative effect of VEGF and SB-431542 on EC sprouting. In vivo, LY-2157299 induced angiogenesis and enhanced VEGF- and basic-fibroblast-growth-factor-induced angiogenesis in a Matrigel-plug assay, whereas adding an alpha5-integrin-neutralizing antibody to the Matrigel selectively inhibited this enhanced response. Thus, induction of alpha5-integrin expression is a key determinant by which inhibitors of TGFbeta type-I receptor kinase and VEGF synergistically promote angiogenesis.

FHL-2 Suppresses VEGF-Induced Phosphatidylinositol 3-Kinase/Akt Activation via Interaction With Sphingosine Kinase-1

In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

56: In vitro generation of human microvessel networks

Introduction: Successful incorporation of bioengineered tissues requires vascularization. Attempts at de novo vascular generation or angiogenic induction have had little success in improving tissue perfusion. An alternate approach to neovascularization is to form a prevascularized construct that is capable of rapid inosculation and rapid cellular perfusion. Human parathyroid (PTH) tissue is uniquely capable of a rapid angiogenic induction, with restoration of calcium homeostasis within 6 weeks of PTH autografting. We hypothesized that human PTH cells could induce human microvessel fragments to form networks of capillary microvessels in vitro. Because PTH cells release VEGF and Angiopoietin-2 (Ang-2) after devascularization, we further hypothesized that PTH angiogenic induction is driven by PTH release of VEGF and Ang-2. Methods: Human subcutaneous fat was partially digested with collagenase to yield microvessel fragments. After sequential filtering, 30-350 micron fragments were embedded in DMEM + 10% FBS-containing collagen I matrix in 3 dimensional culture. PTH cells were cocultured on the matrix surface to induce an angiogenic response. Other matrix cultures were treated with VEGF (50 ng/ml) or VEGF + Ang-2 (50 ng/ml) and microvessel responses noted. Because Stat3 phosphorylation has been identified with endothelial cell (EC) angiogenic responses, we also tested for Stat3 activation in human dermal microvessel EC (HDMEC) after treatment with VEGF or VEGF + Ang-2. Results: Human microvessel fragments in culture are completely quiescent. Coculture with PTH cells induces the rapid development of neovascular networks. Treatment of the human microvessels in vitro with VEGF failed to induce an angiogenic event. VEGF + Ang-2 induced EC migration but no capillary formation. Both VEGF and Ang-2 induced Stat3 phosphorylation in HDMEC, but not in iliac vein EC. Conclusions: Human PTH cells release or induce release of all necessary angiogenic factors for capillary network angiogenic induction of human microvessels in vitro. The mechanism of angiogenic induction is more complex than VEGF stimulation alone. Stat 3 activity is upregulated in microvessel EC after VEGF or Ang-2 stimulation, but not in large vessel EC, suggesting a possible role in microvessel angiogenic processes. PTH cells have the angiogenic potential to induce capillary network development in vitro. This system may be capable of providing vascularization of bioengineered tissues for therapeutic intervention.

Aspirin-triggered Lipoxin A4 inhibition of VEGF-induced endothelial cell migration involves actin polymerization and focal adhesion assembly

Angiogenesis, the growth of new capillaries from pre-existing ones, occurs through dynamic functions of the endothelial cells (EC), including migration, which is essential to achieve an organized formation of the vessel sprout. We demonstrated previously that an aspirin-triggered lipoxin analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1), inhibits vascular endothelial growth factor (VEGF)-induced EC migration. In the present study, we investigated the effects of ATL-1 in the actin cytoskeleton reorganization of EC stimulated with VEGF. Pretreatment of EC with ATL-1 caused a reduction in VEGF-induced stress fibers and therefore reduced the intracellular content of filamentous actin. A concomitant impairment in stress-activated protein kinase (SAPK2/p38) phosphorylation suggests that ATL inhibition of VEGF-stimulated actin polymerization involves the SAPK2/p38 pathway. Moreover, ATL-1 treatment inhibited focal adhesion clustering due to inhibition of focal adhesion kinase (FAK) phosphorylation and the subsequent association of FAK with the actin cytoskeleton. This final event, which ultimately allows cell migration, was reverted by an LX receptor antagonist, but not by a cys-LT1R antagonist, indicating an effect via the G-protein-linked LXA4 receptor. Together our results provide evidence that ATL-1 inhibits EC migration via the concerted inhibition of actin polymerization and proper assembly of focal adhesions, supporting a role for these novel lipid mediators as angiogenesis modulators.

Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells.

Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the proliferation, differentiation, and migration of a large variety of cell types. Like other members of the transforming growth factor-beta family, BMPs elicit their cellular effects through activating specific combinations of type I and type II serine/threonine kinase receptors and their downstream effector proteins, which are termed Smads. In the present study, we investigated BMP receptor/Smad expression and signaling in endothelial cells (ECs) and examined the effects of BMP on EC behavior. Immunohistochemical analysis of tissue sections of human colon and mouse heart and aorta showed that BMP receptors are expressed in ECs in vivo. Bovine aortic ECs and mouse embryonic ECs were found to express BMP receptors and their Smads. BMP receptor activation induced the phosphorylation of specific Smad proteins and promoted EC migration and tube formation. Id1 was identified as a BMP/Smad target in ECs. Ectopic expression of Id1 mimicked BMP-induced effects. Importantly, specific interference with Id1 expression blocked BMP-induced EC migration. The BMP/Smad pathway can potently activate the endothelium. Id1 expression is strongly induced by BMP in ECs. Ectopic expression of Id1 induces EC migration and tube formation. Moreover, Id1 played a critical role in mediating BMP-induced EC migration.

Leptin induces endothelial cell migration through Akt, which is inhibited by PPARgamma-ligands.

Migration of endothelial cells (EC) is a key event in angiogenesis that contributes to neovascularization in diabetic vasculopathy. Leptin induces angiogenesis and is elevated in obesity and hyperinsulinemia. The antidiabetic thiazolidinediones (TZD) inhibit leptin gene expression and vascular smooth muscle cell migration through activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma). This study investigates the role of leptin in EC migration, the chemotactic signaling pathways involved, and the effects of the TZD-PPARgamma ligands troglitazone (TRO) and ciglitazone (CIG) on EC migration. We demonstrate that leptin induces EC migration. Because activation of two signaling pathways, the phosphatidylinositol-3 kinase (PI3K)-->Akt-->eNOS and the ERK1/2 MAPK pathway, is known to be involved in cell migration, we used the pharmacological inhibitors wortmannin and PD98059 to determine if chemotactic signaling by leptin involves Akt or ERK1/2, respectively. Both wortmannin and PD98059 significantly inhibited leptin-induced migration. Treatment with the TZD-PPARgamma-ligands TRO and CIG significantly inhibited the chemotactic response toward leptin. Both PPARgamma-ligands inhibited leptin-stimulated Akt and eNOS phosphorylation, but neither attenuated ERK 1/2 activation in response to leptin. The inhibition of Akt-phosphorylation was accompanied by a PPARgamma-ligand-mediated upregulation of PTEN, a phosphatase that functions as a negative regulator of PI3K-->Akt signaling. These experiments provide the first evidence that activation of Akt and ERK 1/2 are crucial events in leptin-mediated signal transduction leading to EC migration. Moreover, inhibition of leptin-directed migration by the PPARgamma-ligands TRO and CIG through inhibition of Akt underscores their potential in the prevention of diabetes-associated complications.

CD40 ligand inhibits endothelial cell migration by increasing production of endothelial reactive oxygen species.

The CD40/CD40 ligand system is involved in atherogenesis. Activated T lymphocytes and platelets, which express high amounts of CD40 ligand (CD40L) on their surface, contribute significantly to plaque instability with ensuing thrombus formation, leading to acute coronary syndromes. Because reendothelialization may play a pivotal role for plaque stabilization, we investigated a potential role of CD40L on endothelial cell (EC) migration. Stimulation of ECs with recombinant CD40L prevented vascular endothelial growth factor (VEGF)-induced EC migration, as determined by a "scratched wound assay." In addition, activated T lymphocytes and platelets significantly inhibited VEGF-induced EC migration and tube formation in vitro. Because the activation of endothelial nitric oxide (NO) synthase and the release of NO are required for EC migration and angiogenesis, we analyzed the effect of NO. Coincubation with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) did not reverse the inhibitory effect of CD40L on VEGF-induced EC migration and tube formation. In addition, EC migration induced by SNAP was completely inhibited by CD40L. CD40L, however, induced the production of reactive oxygen species and reduced endothelial NO bioavailability. This reactive oxygen species-dependent effect of CD40L stimulation was reversed with vitamin C or N-acetylcysteine. The activation of the CD40 receptor inhibits EC migration by increasing reactive oxygen species. The blockade of EC migration by CD40L may critically affect endothelial regeneration after plaque erosion and thereby may contribute to the increased risk for development of acute coronary events in patients with high circulating levels of CD40L.

Secreted phospholipase A2 induces vascular endothelial cell migration

Secreted phospholipase A2 (sPLA2) regulates a variety of cellular functions. The present investigation was undertaken to elucidate the potential role of sPLA2 in endothelial cell (EC) migration. Bovine aortic endothelial cells (BAECs) exposed to sPLA2 placed in the lower compartment of a modified Boyden chamber displayed increased migration compared to cells exposed to vehicle. The effect of sPLA2 on EC migration was time and dose dependent. Migration of BAECs was observed at 30 minutes, increased over 1 to 2 hours, and declined thereafter. At 2 hours of stimulation, sPLA2 (0.01-2 μmol/L) induced 1.2- to 3-fold increased cell migration compared with media alone. Among the different sPLA2s tested, bee venom, Naja naja, and porcine and human pancreatic PLA2s all evoked a migratory response in ECs. Moreover, human synovial fluid, obtained from patients with arthritis and containing sPLA2 activity, induced EC migration. Migration of ECs was significantly reduced after exposure to a catalytic site mutant of pancreatic sPLA2with decreased lipolytic activity as compared to wild-type sPLA2. Similarly, pretreatment of human synovial fluid withp-bromophenacyl bromide, an irreversible inhibitor of sPLA2, markedly decreased the ability of human synovial fluid to stimulate EC migration. Moreover, migration of ECs was stimulated on exposure to hydrolytic products of sPLA2activity including arachidonic acid, lysophosphatidic acid, and lysophosphatidylcholine. These findings suggest that sPLA2plays a physiologic role in induction of EC migration. Moreover, the effects of sPLA2 on EC migration are mediated, at least in part, by its catalytic activity.