Early Stages Of Angiogenesis Research Articles

Temporal and pharmacological characterization of angiostatin release and generation by human platelets: implications for endothelial cell migration.

Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF). Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo-vascularization following thrombus formation.

Low molecular mass dermatan sulfate modulates endothelial cells proliferation and migration

Low molecular mass dermatan sulfate, obtained by depolymerization, induced the entrance in S phase of mitosis, enhanced the activity of matrix metalloproteinase-2, and could modulate cell migration of endothelial cells, through mechanisms independent of TNF-α autocrine regulation. LMMDS located at the injured sites could influence early stages of angiogenesis.

Imaging Cancer's Blood Supply

In the earliest stages of angiogenesis, vascular endothelial cells respond to chemical signals and start to proliferate. Before long, the cells send the integrin αvβ3 and the platelet endothelial cell adhesion molecule CD31/PECAM onto their outer membranes like tiny flags. Even though the endothelial cells have not become polarized (oriented in a certain direction), the αvβ3 molecules are expressed within what will eventually become the lumen of the vessel and the outside of the vessel wall. Researchers aiming to trace the origins of angiogenesis for heart disease or cancer start in the same place because each of these diseases depends on the same blood vessel formation process.

Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering

We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young’s moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis.

Challenging the Surgical Rodent Hindlimb Ischemia Model with the Miniinterventional Technique

Purpose To develop an interventional hindlimb ischemic model and compare its angiogenic effect versus surgical ligation (SL) and excision of the femoral artery in rats treated with transplantation of bone marrow mononuclear cells (MNCs) as an angiogenic stimulator. Materials and Methods Forty-eight Lewis rats randomly received interventional embolization (IE) with hydrogel wire or SL and excision of the right femoral artery. Rodents were intraarterially transplanted with 1.5 × 10 7 MNCs in 500 μL medium from 24 isogenic donor rats. Functional and structural recovery was evaluated by laser Doppler imaging (LDI), cytokine/chemokine assay, and histologic staining. Results In vivo microscopic images showed significantly dilated vasa vasorum around the embolized segment of the right femoral artery at 3 days compared with disorganized tissue structure in the SL group. However, the LDI index was significantly higher in the SL group at 3 days compared with the IE group. LDI did not significantly differ between the two groups at 2 weeks after transplantation. Cytokine assay showed higher levels of interleukin (IL)–1α and IL-18 in the SL group; the IE group had higher levels of interferon-γ, IL-6, IL-13, and granulocyte colony-stimulating factor. Histologic examination demonstrated inflammatory infiltration near the incision within nerve fibers with dilated capillaries, showing nerve degeneration in the SL group. At 2 weeks, histologic analysis demonstrated massive scarring under the skin spreading into the musculature in the SL group. Conclusions A minimally invasive hindlimb ischemia model has been successfully developed that preserves tissue integrity and minimizes inflammation and confounding factors in the early stages of angiogenesis and arteriogenesis.

Analysis of vascular distribution and growth factors in human gingival tissue associated with periodontal probing depth

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel endothelium. Tissue levels of this angiogenesis marker are unknown in human gingival tissue, as is the correlation between vascular growth factors and hypoxia-inducible factor. We examined the expression of VEGF, type III tyrosine kinase receptors (VEGF-R2), platelet-endothelial cell adhesion molecule (CD31) and hypoxia-inducible factor (HIF) mRNA from human gingival tissue of the oral cavity. Tissue samples were from a small quantity of gingival sample biopsy with gingival sulcular depth (GSD) < 2 mm (Group 1), 2 to 4 mm (Group 2), and > 4 mm (Group 3). We found that the levels of VEGF-R2, CD31 and HIF mRNA were higher in the gingival tissue of Group 2 than that of Group 1, and VEGF in the Group 3 was also higher than that of Group 1. The different mRNA levels of these markers may reflect the mRNA levels reflect the vasculature state of gingival tissue based on GSD. VEGF-R2 and HIF also indicate the presence of an elongated blood vessel in the gingival tissue. In the early stage of angiogenesis, VEGF-R2 leads to expression of VEGF, and HIF-1 mediates increased VEGF expression in response to hypoxia in swollen tissues or during the expansion of periodontal tissues, which is useful in the early diagnosis of periodontal diseases.

Visualizing vascular permeability and lymphatic drainage using labeled serum albumin

During the early stages of angiogenesis, following stimulation of endothelial cells by vascular endothelial growth factor (VEGF), the vascular wall is breached, allowing high molecular weight proteins to leak from the vessels to the interstitial space. This hallmark of angiogenesis results in deposition of a provisional matrix, elevation of the interstitial pressure and induction of interstitial convection. Albumin, the major plasma protein appears to be an innocent bystander that is significantly affected by these changes, and thus can be used as a biomarker for vascular permeability associated with angiogenesis. Traditionally, albumin leak in superficial organs was followed by colorimetry or morphometry with the use of albumin binding vital dyes. Over the last years, the introduction of tagged-albumin that can be detected by various imaging methods, such as magnetic resonance imaging and positron emission tomography, opened new possibilities for quantitative three dimension dynamic analysis of permeability in any organ. Using these tools it is now possible to follow not only vascular permeability, but also interstitial convection and lymphatic drain. Active uptake of tagged albumin by caveolae-mediated endocytosis opens the possibility for using labeled albumin for vital staining of cells and cell tracking. This approach was used for monitoring recruitment of perivascular stroma fibroblasts associated with tumor angiogenesis.

Abstract 5218: γ-secretase inhibitor PF-03084014 impairs Notch signaling and induces antiangiogenic and antitumor effects in breast cancer models

Abstract PF-03084014, a small molecule γ-secretase inhibitor (GSI), has previously been shown to inhibit Notch signaling and demonstrated antitumor efficacy. The aim of this report is to gain insights into the mechanism PF-03084014-induced activity. The cell-based evaluation of PF-03084014 across 30 breast cancer (BC) cell lines indicated significant growth inhibition in only a subset of cell lines (3/30). In contrast, PF-03084014 demonstrated cell cycle arrest and induction of apoptosis by FACS analysis across the majority of a panel of T-acute lymphoblastic leukemia (T-ALL) cell lines. In vivo BrdU uptake of tumor and [18F]FLT-PET imaging studies demonstrated that PF-03084014 at an efficacious dose (120 mg/kg) caused cell cycle arrest in Sup T1 tumors but not in MDA-MB-231 xenografts, although PF-03084014 suppressed the expression of Notch target genes and tumor growth in both models. These results suggest that the primary mechanism(s) for PF-03084014-induced efficacy in T-ALL and BC models may be different, possibly because the tumor-host microenvironment in the BC model plays an important role during Notch signaling activation. IHC analysis in a panel of solid tumors demonstrated that Notch ligands including jagged 1, 2 and Dll4 were predominately expressed in the host vasculature and stromal cells. To investigate the potential antiangiogenic activity of PF-03084014, an in vitro endothelial cell/fibroblast co-culture tube formation assay was utilized. PF-03084014 disrupted the multicellular lumen-like structures at 100 nM, indicating defective differentiation of endothelial cells in early stage angiogenesis. Ultrasound imaging was utilized to further investigate the antiangiogenic properties of PF-03084014 including both the quantitative measurement of the total tumor vessel volume using 3-D scanning methods and an assessment tumor blood vessel function by measuring blood flow after iv injection of microbubble contrast agents. When MDA-MB-231 tumor bearing mice were administered with an efficacious dose of PF-03084014 consecutively for 4 days, ultrasound imaging revealed a significant (p&amp;lt; 0.05) decrease of total tumor vessel volume and function compared to the vehicle treatment. A subsequent FITC-lectin vascular perfusion assay was performed to confirm the reduction in functional microvasculature by PF-03084014. In addition to the antiangiogenic effect, we also observed treatment-induced caspase 3 activation and Ki67 suppression in the MDA-MB-231 tumor model (2 days after treatment), suggest the GSI also induces apoptosis and cytoreductive activity. Collectively, our data provide insights that activation of Notch signaling requires the interaction of tumor cells with their host environment in solid tumors, and impairing Notch signaling by PF-03084014 results in significant antitumor efficacy in the breast cancer xenografts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5218.

Rapamycin Inhibition of the Akt/mTOR Pathway Blocks Select Stages of VEGF-A 164 –Driven Angiogenesis, in Part by Blocking S6Kinase

We evaluated the stages of VEGF-A(164) driven angiogenesis that are inhibited by therapeutic doses of rapamycin and the potential role of S6K1 in that response. We assessed the effects of rapamycin on the several stages of angiogensis and lymphangiogenesis induced with an adenovirus expressing VEGF-A(164) (Ad-VEGF-A(164)) in the ears of adult nude mice. Rapamycin (0.5 mg/kg/d) effectively inhibited mTOR and downstream S6K1 signaling and partially inhibited Akt signaling, likely through effects on TORC2. The earliest stages of angiogenesis, including mother vessel formation and increased vascular permeability, were strikingly inhibited by rapamycin, as was subsequent formation of daughter glomeruloid microvasular proliferations. However, later stage formation of vascular malformations and lymphangiogenesis were unaffected. Retrovirally delivered isoforms and shRNAs demonstrated that S6K1 signaling plays an important role in early VEGF-A(164)-angiogenesis. Rapamycin potently inhibited early and mid stages of VEGF-A(164)-driven angiogenesis, but not late-stage angiogenesis or lymphangiogenesis. Rapamycin decreased phosphorylation of both Akt and S6, suggesting that both the TORC1 and TORC2 pathways are impacted. Inhibition of S6K1 signaling downstream of mTOR is a major component of the antiangiogenesis action of rapamycin.

Pericytes promote angiogenesis at the early stage of wound healing

Recently, more and more attention has been focused on pericytes as a target of anti‐angiogenic therapy. In this study, we sought to determine whether they exist at the early stage of angiogenesis, and if they existed, determine how they function at this stage during wound healing. In wound tissue, NG2 proteoglycan and desmin immunoreactive pericytes were closely associated with newly forming blood vessels that contained endothelia with BrdU incorporated proliferative cell nuclei. These pericytes co‐expressed vascular endothelial growth factor ‐A (VEGF‐A). Association of pericytes on the migrating/sprouting endothelia with mitotic figure was also observed with transmission electron microscopy. Phenotypically, the pericytes in wound tissues had larger cell bodies and more euchromatic nuclei than the pericytes in normal vasculature, and expressed alpha smooth muscle actin in general, which is unusual phenomenon especially in normal capillary pericytes. These observations together indicate that the activated state of pericytes is essential for the angiogenesis at the early stage of wound healing.We conclude that pericytes do exist at the early stage of the angiogenesis during wound healing to induce proliferation of endothelia and to guide their migration/sprouting via VEGF‐A secretion, though the generally accepted view suggests the absence or disappearance of pericytes at this stage of angiogenesis.

056 Activation of Sterol Regulatory Element-Binding Proteins ((SREBPs) is Critical in IL-8-Induced Angigiogenesi

Angiogenesis is an essential process during wound healing. The chemokine IL-8 is a well-known inflammatory and angiogenic factor. We found that IL-8 stimulates activation of SREBPs, molecules that are critical for cholesterol and fatty acid metabolism, and that this activation is markedly increased during IL-8-induced angiogenesis. Two important processes in the early stages of angiogenesis are increase in proliferation and migration of endothelial cells. Therefore, we tested whether IL-8 stimulates these processes in a SREBP-dependent manner both in vivo and in culture. Our data show that IL-8 stimulates SREBP-1 and -2 gene expression which leads to an increase in both the precursor and the mature forms of these proteins in human microvascular endothelial cells (hMVEC). In addition, several genes downstream of SREBPs are also up-regulated by IL-8 stimulation, in particular 3-hydroxy -3-methylglutaryl coenzyme A reductase (HMGCA). Furthermore, SREBP-1 was abundant in the neovasculature of wound healing skin, and this expression could be suppressed by local application of antibodies to IL-8 or to its receptors, CXCR1 and CXCR2. To test whether SREBP activation is critical for the proliferation and migration of endothelial cells induced by IL-8 during angiogenesis, we used either RNA interference (RNAi) to inhibit the SREBP cleavage-activating protein (SCAP) that escorts SREBPs from the ER to the Golgi for maturation (activation), or treated endothelial cells with 25-hydroxycholesterol (25-HC), an inhibitor of SREBP activation. Both SCAP RNAi and 25-HC significantly inhibited IL-8-induced SREBP activation during endothelial cell migration and proliferation indicating that SREBPs are critical components of the signaling pathways leading to IL8-induced angiogenesis. In addition, in the chick chorioallantoic membrane (CAM) model, 25-HC markedly inhibited both developmental- and IL-8-induced angiogenesis in 7-day and 10-day CAMs, respectively. In conclusion, our results indicate a novel regulatory mechanism for IL-8-induced angiogenesis that is mediated by SREBPs.

Assessment of vascular endothelial growth factor expression and apoptosis in the ovarian graft: can exogenous gonadotropin promote angiogenesis after ovarian transplantation?

To evaluate the effects of gonadotropin on angiogenesis by assessing vascular endothelial growth factor (VEGF) expression in rat ovaries transplanted after freezing and thawing. In vitro laboratory experiments. Academic research institute. Sixty immature female rats. Frozen-thawed ovaries were autotransplanted into the SC tissue of 60 rats (ages between 21 and 28 days). After transplantation, either pregnant mare's serum gonadotropin (PMSG) or saline was administered. The grafted ovaries were collected 2, 7, and 30 days after transplantation for evaluation. Assessment of the morphology and number of follicles, evaluation of apoptosis, and analysis of VEGF expression in the grafted ovaries. Most follicles in the grafts were apoptotic on day 2 but recovered by day 7. The proportion of antral follicles and corpora lutea in the graft correlated with the duration after transplantation. A significant increase in the expression of VEGF188 mRNA was noticed in the grafted ovaries on day 2. Moreover, the mRNA expression in the PMSG group was higher than that in the control group. The increased VEGF protein production in the graft was confirmed by Western blot analysis. In ovariectomized animals, gonadotropin treatment may not provide any added benefits for folliculogenesis and angiogenesis. Nevertheless, a significant increase in the VEGF188 isoform in the gonadotropin-treated group may suggest the positive effect of exogenous gonadotropin therapy in the early stages of angiogenesis.

Interaction of Estrogen and Tumor Necrosis Factor α in Endothelial Cell Migration and Early Stage of Angiogenesis

The role of estrogen replacement therapy in postmenopausal women remains controversial. The authors hypothesized that contradictory results with estrogen therapy may be explained by estrogen's potent proangiogenic property, which could be protective in women without atherosclerotic disease but in the presence of chronic inflammation, could lead to destabilization of atherosclerotic plaques. The authors thus examined the interaction between 17beta-estradiol (E2) and the inflammatory cytokine tumor necrosis factor alpha (TNFalpha) in an early stage of angiogenesis. Human umbilical endothelial cells were grown to confluence. Migration was assessed with a wound assay and proliferation was assessed with 5-bromo-2'-deoxyuridine (BrDU). Cells were treated with medium alone, TNFalpha at 0.3, 1, or 20 ng/ml, E2 at 20 nM, or the combination of E2 and TNFalpha. The authors used real-time polymerase chain reaction (PCR) to measure changes in expression of the angiogenesis genes angiopoeitin-2 (Ang-2), vacular endothelial growth factor (VEGF)-A and -C, and interleukin (IL)-8. A large dose of TNFalpha (20 ng/ml) inhibited healing at 24 to 48 h and the addition of E2 preserved some healing. E2 by itself doubled migration, with only a minimal effect on proliferation. A low dose of TNFalpha (0.3 ng/ml) had no effect on migration, 1.0 ng/ml moderately increased it, but the addition of E2 to both doses of TNFalpha increased migration. There was no change in migration when cells were pretreated with E2 and given TNFalpha after wounding, whereas pretreatment with TNFalpha followed by E2 significantly increased wound healing. The nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine-methyl ester (l-NAME) completely blocked the E2 effect on migration. TNFalpha (0.3 and 1.0 ng/ml) increased expression of VEGF-C (2.8 +/- 0.1- and 2.5 +/- 0.2-fold, respectively) and IL-8 (32.8 +/- 1.2- and 42.7 +/- 3.6-fold, respectively) mRNA, but E2 had no significant effect on these molecules. E2 increases the angiogenic activity of TNFalpha. This could potentially worsen the stability of complex atherosclerotic plaques and increase cardiovascular events.

Transactivation of vascular endothelial growth factor receptor-2 by interleukin-8 (IL-8/CXCL8) is required for IL-8/CXCL8-induced endothelial permeability.

Interleukin-8 (IL-8/CXCL8) is a chemokine that increases endothelial permeability during early stages of angiogenesis. However, the mechanisms involved in IL-8/CXCL8-induced permeability are poorly understood. Here, we show that permeability induced by this chemokine requires the activation of vascular endothelial growth factor receptor-2 (VEGFR2/fetal liver kinase 1/KDR). IL-8/CXCL8 stimulates VEGFR2 phosphorylation in a VEGF-independent manner, suggesting VEGFR2 transactivation. We investigated the possible contribution of physical interactions between VEGFR2 and the IL-8/CXCL8 receptors leading to VEGFR2 transactivation. Both IL-8 receptors interact with VEGFR2 after IL-8/CXCL8 treatment, and the time course of complex formation is comparable with that of VEGFR2 phosphorylation. Src kinases are involved upstream of receptor complex formation and VEGFR2 transactivation during IL-8/CXCL8-induced permeability. An inhibitor of Src kinases blocked IL-8/CXCL8-induced VEGFR2 phosphorylation, receptor complex formation, and endothelial permeability. Furthermore, inhibition of the VEGFR abolishes RhoA activation by IL-8/CXCL8, and gap formation, suggesting a mechanism whereby VEGFR2 transactivation mediates IL-8/CXCL8-induced permeability. This study points to VEGFR2 transactivation as an important signaling pathway used by chemokines such as IL-8/CXCL8, and it may lead to the development of new therapies that can be used in conditions involving increases in endothelial permeability or angiogenesis, particularly in pathological situations associated with both IL-8/CXCL8 and VEGF.

Adhesive and Proteolytic Phenotype of Migrating Endothelial Cells Induced by Thymosin β‐4

The early stages of angiogenesis are usually accompanied by the occurrence of vascular leakage, and the deposition of fibrin in extravascular spaces. Initially, the fibrin network acts as a sealing matrix, but later on also as a scaffolding for invading endothelial cells. This process is induced by angiogenic growth factors, particularly by vascular endothelial growth factor (VEGF). Angiogenesis involves proteolytic activities, in particular cell-bound urokinase/plasmin and matrix metalloproteinase (MMPs) activities that modulate the fibrin structure and affect adhesion and migration of endothelial cells. Recent data show that formation of new vessels may be stimulated by thymosin beta-4 (Tbeta-4), but it is still not clear whether Tbeta-4 alone is angiogenic or the angiogenic potential of Tbeta-4 is mediated by VEGF. In this report to further characterize Tbeta-4 angiogenic activity, we produced its mutants that were deprived of the N-terminal tetrapeptide AcSDKP (Tbeta-4((AcSDKPT/4A))), the actin-binding sequence KLKKTET (Tbeta-4((KLKKTET/7A))) and with the nuclear localization sequence damaged by a point mutation Lys16Ala (Tbeta-4((K16A))). Then we tested their activity to induce expression and release of MMPs as well as plasminogen activators inhibitor type-1 (PAI-1). We also analyzed their effect on migration and proliferation of endothelial cells in three-dimensional (3D) fibrin matrix as well as on their ability to stimulate the outgrowth of human endothelial cells in capillary-like tubular structures. Our data demonstrate that increased intracellular expression of Tbeta-4 and its mutants is necessary and sufficient to induce PAI-1 gene expression in endothelial cells. Similarly, they stimulate expression and release of MMP-1, -2, and -3. As evaluated by using specific inhibitors to these MMPs, they modified specifically the structure of fibrin and thus facilitated migration of endothelial cells. To sum up, our data show that the mechanism by which Tbeta-4 induced transition of endothelial cells from quiescent to proangiogenic phenotype is characterized by increased expression of PAI-1 and MMPs did not require the presence of the N-terminal sequence AcSDKP, and depended only partially on its ability to bind G-actin or to enter the nucleus.

Arsenic Trioxide Affects Early Stage Angiogenesis through Inhibition of CD14 Monocyte Transdifferentiation into Endothelial Cells Induced by Pleiotrophin and mCSF.

We have discovered a novel mechanism leading to blood vessel formation involving transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (H. Chen et al, Blood, 2005; Yeh et al BJH, 2006). Arsenic trioxide (ATO) induces apoptosis of cancer cells directly through a number of mechanisms, and this drug has also been shown to inhibit angiogenesis. However, it remains unknown whether ATO affects the earliest stages of angiogenesis and vasculogenesis important in tumor development. We purified human monocytes (CD14+) and cultured these cells on collagen I-coated dishes. mCSF was added to the cells after 1 hour of culture. PTN was added twice to the culture, once after 24 hours and again after 5 days with or without ATO or bortezomib. FLK-1 expression (VEGFR-2) showed that the cells incubated on collagen I without drugs formed tube-like structures in the presence of PTN and mCSF. However, the tube-like structures disappeared after adding either the IC50 (5x10−6M) dose or low (5x10−7M) dose of ATO. FLK-1 staining remains in the tube-like structures with low doses (3x10−12M) of bortezomib. In order to examine whether ATO or bortezomib affects endothelial gene expression when monocytes are induced to transdifferentiate in the presence of these cytokines, we also examined expression using RT-PCR on endothelial cell genes (vascular endothelial growth factor receptor-2 (Flk-1), Tie-2 and von Willebrand factor (vWF)) and Western blot analysis for protein expression. The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at both the higher (5x10−6M) and lower (5x10−7M) doses of ATO. In contrast, the expression of endothelial markers was not reduced by adding low dose bortezomib (3x10−12M). We further examined the effects of ATO and bortezomib on early stage angiogenesis in vivo using the chorioallantoic membrane (CAM) assay. Fertilized chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. The tested micro-sponge with ATO (5x10−6M) or bortezomib (3x10−11M) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubator for 48 hours. The assay scored positive when two independent observers reported a significant reduction of vessels in the treated area. The results of the CAM assay showed that compared to saline, ATO significantly reduced new macroscopic and microscopic vessel formation. In contrast, bortezomib did not affect angiogenesis in the CAM assay. These experiments define a previously unrecognized novel mechanism by which ATO may have anti-angiogenetic effects in cancer patients-preventing the transdifferentiation of monocytes into endothelial cells by PTN. They also suggest ATO as a potential new specific agent to inhibit angiogenesis resulting from transdifferentiation of monocytes into vascular endothelial cells driven by pleiotrophin and mCSF. These results suggest a novel way by which anti-cancer agents may impact angiogenesis.

Carcinoembryonic antigen-related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1-/- mice, the Ceacam1 gene was deleted systemically, and in CEACAM1(endo+) mice, CEACAM1 was overexpressed under the control of the endothelial cell-specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1-/- mice failed to establish new capillaries whereas in CEACAM1(endo+) mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1-/- mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1(endo+) mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease.

JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA and protein in response to actin cytoskeleton reorganization in endothelial cells

Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF-induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP.

Differential gene expression during capillary morphogenesis in a microcarrier-based three-dimensional in vitro model of angiogenesis with focus on chemokines and chemokine receptors.

To globally compare the gene expression profiles during the capillary morphogenesis of human microvascular endothelial cells (HMVECs) in an in vitro angiogenesis system with affymetrix oligonucleotide array. A microcarrier-based in vitro angiogenesis system was developed, in which ECs migrated into the matrix, proliferated, and formed capillary sprouts. The sprouts elongated, branched and formed networks. The total RNA samples from the HMVECs at the selected time points (0.5, 24, and 72 h) during the capillary morphogenesis were used for microarray analyses, and the data were processed with the softwares provided by the manufacturers. The expression patterns of some genes were validated and confirmed by semi-quantitative RT-PCR. The regulated genes were grouped based on their molecular functions and expression patterns, and among them the expression of chemokines and chemokine receptors was specially examined and their functional implications were analyzed. A total of 1 961 genes were up- or down-regulated two-folds or above, and among them, 468 genes were up- or down-regulated three-folds or above. The regulated genes could be grouped into categories based on their molecular functions, and were also clustered into six groups based on their patterns of expression. As for chemokines and chemokine receptors, CXCL1/GRO-alpha, CXCL2/GRO-beta, CXCL5/ENA-78, CXCL6/GCP2, IL-8/CXCL8, CXCL12/SDF-1, CXCL9/Mig, CXC11/ITAC, CX3CL1/fractalkine, CCL2/MCP-1, CCL3, CCL5/RANTES, CCL7, CCL15, CCL21, CCL23, CCL28, and CCR1, CCR9, CXCR4 were identified. Moreover, these genes demonstrated different changing patterns during the capillary morphogenesis, which implied that they might have different roles in the sequential process. Among the chemokines identified, CCL2/MCP-1, CCL5/RANTES and CX3CL1 were specially up-regulated at the 24-h time point when the sprouting characterized the morphological change. It was thus suggested that they might exert crucial roles at the early stage of angiogenesis. The present study demonstrates a global profile of gene expression during endothelial capillary morphogenesis, and the results provide us much information about the molecular mechanisms of angiogenesis, with which further evaluation of individual genes can be conducted.

Characterization of initiation of angiogenesis in early stages of prostate adenocarcinoma development and progression in a transgenic murine model

Objectives A novel prostate-specific protein of 94 amino acids (PSP94) promoter/enhancer-directed transgenic murine model of prostate cancer (CaP) was examined to determine the temporal relationship of angiogenesis with the development and progression of CaP. Methods Immunohistochemistry and in situ hybridization were performed to determine the protein and mRNA expression of basic fibroblast growth factor, vascular endothelial growth factor, and microvessel formation at different stages of CaP development and progression. Results Differential expression of basic fibroblast growth factor and vascular endothelial growth factor protein was found with the development and progression of CaP ( P <0.0001 and P <0.0001, respectively). Angiogenesis was identified in CaP. mRNA expression of basic fibroblast growth factor and vascular endothelial growth factor was present from high-grade prostatic intraepithelial neoplasia to poorly differentiated CaP. Conclusions The PSP94 gene is conserved in humans and rodents; therefore, the PSP94 transgenic murine model of CaP provides a preclinical model to test such antiangiogenic or other chemopreventive therapies that may be predictive of human CaP.