Proliferation Of Microvascular Endothelial Cells Research Articles

Angiostatic activity of obtustatin as α1β1 integrin inhibitor in experimental melanoma growth

The presented results show the effect of targeting of collagen receptor, alpha1beta1 integrin expressed on the endothelial cells on the development of experimental melanoma and pathological angiogenesis. Obtustatin, a snake venom KTS-disintegrin, was applied as a specific inhibitor of this integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression in 2 animal systems, mouse and quail. Its oncostatic effect was related to the inhibition of angiogenesis. Obtustatin inhibited the neovascularization ratio on the CAM embryo of quail, which was pathologically induced by the developing tumor. The i.v. administration of obtustatin completely blocked cancer growth of MV3 human melanoma in nude mice. In B16F10 syngeneic mouse model treatment with the disintegrin revealed a lower effect, although the development of the tumor was significantly reduced for both dosages. The mechanism of obtustatin action is related to the blocking of microvascular endothelial cell proliferation, which undergoes apoptosis in caspase-dependent manner. Summarizing, we present studies of low molecular weight disintegrin, obtustatin as a potential therapeutic compound for treatment of melanoma that contain a high level of vascularization.

Lysophosphatidic Acid Mediates Interleukin-8 Expression in Human Endometrial Stromal Cells through Its Receptor and Nuclear Factor-κB-Dependent Pathway: A Possible Role in Angiogenesis of Endometrium and Placenta

Lysophosphatidic acid (LPA) is a pleiotropic phospholipid molecule involved in inflammation, angiogenesis, would healing, and cancer invasion. Whereas serum lysophospholipase D activity increases in women with pregnancy, the role of LPA in pregnancy remains unclear. We investigated the expression of LPA receptors and function of LPA in endometrial stromal cells. Histologically normal endometrium was obtained from surgical specimens of women undergoing hysterectomy for leiomyoma. First-trimester decidua was obtained from women receiving elective termination of pregnancy. We examined the expressions of LPA1, LPA2, and LPA3 receptors in endometrial stromal cells. The effects of LPA on the expression of vascular endothelial growth factor, IL-6, and IL-8 were examined. Signal pathways of LPA were delineated. Functions of secretory angiogenic factors were tested using human endometrial microvascular endothelial cells. Immunoreactivity and mRNA of LPA1 receptors were identified in endometrial stromal cells. LPA enhanced IL-8 expression in a dose- and time-dependent manner, whereas vascular endothelial growth factor or IL-6 expression was not affected by LPA treatment. Mechanistic dissection disclosed that LPA functioned via the Gi protein, MAPK/p38 and nuclear factor-kappaB pathway. LPA-induced IL-8 enhanced migration, permeability, capillary tube formation, and proliferation of human endometrial microvascular endothelial cells. Endometrial stromal cells express LPA1 receptors. Through the LPA1 receptor, LPA induces IL-8 expression via a nuclear factor-kappaB-dependent signal pathway. These results could suggest that LPA may play a role in angiogenesis of endometrium and placenta through induction of IL-8 in endometrial stromal cells during pregnancy.

Role of αv integrin in osteoprotegerin-induced endothelial cell migration and proliferation

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor κB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin αvβ3 or integrin αvβ5 blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-αvβ3 antibody or anti-αvβ5 antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin αv activity. Taken together, these results suggest that integrins αvβ3 and/or αvβ5 contribute to endothelial cell proliferation and migration induced by OPG.

In-vitro-Untersuchungen zum Wirkungsmechanismus von VEGF und seinen Inhibitoren

VEGF expression and signalling are deregulated in diabetic retinopathy. Cellular processes like migration and proliferation as well as control of the permeability of the endothelium by VEGF are regulated as a consequence of its binding to the VEGF receptor 2. Proteins forming tight junctions between microvascular endothelial cells of the retina are delocated to the cytoplasm after treatment with VEGF(165), likely leading to the breakdown of the blood-retina barrier. VEGF-inhibitors such as a VEGF-aptamer (modified RNA-oligonucleotide; pegaptanib) or specific antibodies/antibody fragments (bevacizumab, ranibzumab) which directly interfere with the interaction of VEGF with its receptors are considered to be promising novel therapeutics to treat diabetic retinopathy and age-related macular degeneration. In vitro studies using microvascular endothelial cells will help to clarify the mechanisms of action of VEGF and its inhibitors. In particular, the influence of VEGF isoforms and the inhibitor ranibizumab on the proliferation and migration of bovine retinal microvascular endothelial cells was studied, as well as the rearrangement of tight-junction proteins after treatment of the cells with VEGF(165) and specific inhibitors. In addition to a review of recent publications in the field, primary data from our own studies are presented in this article.

Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1α with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system

Retinal pigment epithelial (RPE) cells and choroidal microvascular endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), and hypoxia plays an important role in CNV formation via hypoxia inducible factor 1 (HIF-1). Our aim was to evaluate the role of HIF-1 in human RPE cells with regard to proliferation, migration and tube formation of CECs under hypoxia. RPE cells were cultured under chemical hypoxia induced by 200 muM CoCl(2), and RNA interference (RNAi) technique was used to knock down HIF-1alpha gene in RPE cells. mRNA and protein expression of HIF-1alpha and VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. Three kinds of coculture models were used to observe the effects of RPE cells transfected by short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1alpha) on the proliferation, migration and tube formation of CECs respectively. Transfection of shRNA-expressing pDNA targeting HIF-1alpha to RPE cells resulted in the knock down of HIF-1alpha gene and reduction of the corresponding mRNA and protein of HIF-1alpha and VEGF under hypoxia. Consequently, the proliferation, migration and tube formation of CECs were significantly inhibited by the knocked-down RPE cells compared with the control in the coculture system. The proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on days 3, 4 and 5 respectively. Migration reduced by 49.6% at 5 h, and tube formation decreased by 40.4% at 48 h. RNAi of HIF-1alpha in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1alpha.

Synergistic effects of methylnaltrexone with 5-fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor–induced angiogenesis

Many patients with cancer receive combinations of drug treatments that include 5-fluorouracil (5-FU) and bevacizumab. Therapeutic doses of 5-FU are often associated with unwanted side effects, and bevacizumab is costly. Therefore, we explored potential agents that can reduce the therapeutic concentration of these drugs. Our data indicate that methylnaltrexone (MNTX), a peripheral antagonist of the mu-opioid receptor, exerts a synergistic effect with 5-FU and bevacizumab on inhibition of vascular endothelial growth factor (VEGF)-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration, two key components in cancer-associated angiogenesis. MNTX inhibited EC proliferation with an IC(50) of approximately 100 nmol/L. Adding 100 nmol/L MNTX to EC shifted the IC(50) of 5-FU from approximately 5 micromol/L to approximately 7 nmol/L. Further, adding 50 ng/mL MNTX shifted the IC(50) of bevacizumab on inhibition of EC migration from approximately 25 to approximately 6 ng/mL. These synergistic effects were not observed with naltrexone, a tertiary mu-opioid receptor antagonist. On a mechanistic level, we observed that treatment of human EC with MNTX, but not naltrexone, increased receptor protein tyrosine phosphatase mu activity, which was independent of mu-opioid receptor expression. Silencing receptor protein tyrosine phosphatase mu expression (small interfering RNA) in human EC inhibited both synergy between MNTX and bevacizumab or 5-FU and increased VEGF-induced tyrosine phosphorylation of Src and p190 RhoGAP with enhanced activation of Akt and the actin cytoskeletal regulatory protein, RhoA, whereas silencing Src, Akt, or RhoA blocked VEGF-induced angiogenic events. Therefore, addition of MNTX could potentially lower the therapeutic doses of 5-FU and bevacizumab, which could improve index.

The Growth Factor Midkine Antagonizes VEGF Signaling In Vitro and In Vivo

Midkine (MDK) is a heparin-binding growth factor involved in growth, survival, migration, and differentiation of various target cells and dysregulation of MDK signaling is implicated in a variety of inflammatory diseases and cancers. Although MDK has been reported to act on endothelial cells and to have proangiogenic effects, the exact role of MDK in angiogenesis is poorly defined. Here, we report that MDK is actually a modulator of angiogenesis and that it can abrogate the vascular endothelial growth factor A (VEGF-A)-induced proliferation of human microvascular endothelial cells in vitro through the downregulation of proangiogenic cytokines and through the upregulation of the antiangiogenic factor, tissue inhibitor of metalloproteinase 2. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and of downstream signaling molecules, such as phosphatidylinositol-3-kinase and mitogen-activated protein kinases, is also impaired. Moreover, MDK downregulates VEGF-A-induced neovascularization and vascular permeability in vivo. We propose a model in which MDK is a new modulator of the VEGF-A-VEGFR-2 axis.

Glucose restriction inhibits glycolytic flux and limits proliferation in lung microvascular endothelial cells

Pulmonary microvascular endothelial cells (PMVECs) exhibit increased glycolytic flux, which may contribute to their highly proliferative nature. We therefore sought to determine whether PMVECs rely on extracellular glucose to sustain glycolysis and growth under normoxic conditions. We performed growth curves and pH measurements using PMVECs and pulmonary artery endothelial cells (PAECs) in different glucose concentrations: High (22 mM), normal (11 mM) and low (5 mM). PMVEC proliferation was greatest in high glucose, and decreased progressively with lower glucose concentrations. In contrast, glucose concentration did not affect PAEC growth. Whereas PMVECs acidified the media in high glucose, they did not acidify the media at low glucose concentrations; PAECs did not acidify the media at any glucose concentration. QRT‐PCR analysis for glucose transporter‐1 (GLUT‐1), hexokinase‐2 (HK‐2), phosphofructokinase (PFK), and lactate dehydrogenase (LDH) revealed that media glucose did not influence upon the expression of these key glycolytic enzymes, suggesting genes responsible for increased glycolysis are constitutively upregulated in PMVECs. The constitutive upregulation of GLUT‐1, HK‐2, PFK, and LDH in PMVECs illustrate the intrinsic preference of these cells to use aerobic glycolysis during proliferation. Thus, glucose availability is a key determinant of growth and acidosis in PMVECs. HL66299.

Hypoxia‐induced proliferation of human pulmonary microvascular endothelial cells depends on epidermal growth factor receptor (EGFR) tyrosine kinase activity

The hallmark of pulmonary hypertension is vascular remodeling, which has recently been postulated to represent a type of malignant transformation. In cancer therapy, EGFR tyrosine kinase has been recognized as a therapeutic target to prevent tumor cell proliferation. We hypothesized that hypoxia would lead to human pulmonary microvascular endothelial cell (hPMVEC) proliferation via EGFR tyrosine kinase activity. hPMVEC were placed in normoxia (20% O2, 5%CO2) or hypoxia (1% O2, 5% CO2) with or without AG1478 added. Western blotting for total EGFR and PCNA, and proliferation assays were done. Protein levels of total EGFR and PCNA were increased in hypoxia compared to normoxia groups. Hypoxic hPMVEC had a 2‐fold greater level of proliferation compared to normoxic controls. This increase in proliferation was prevented by addition of AG1478. Our data demonstrate that EGFR tyrosine kinase activity is critical in promoting proliferation of hPMVEC in hypoxia. We speculate that EGFR tyrosine kinase inhibition represents a novel therapy to prevent vascular remodeling in pulmonary hypertension.

Endothelial cell proliferation is dependent on Nox4‐containing NADPH oxidases whereas Nox2 is anti‐apoptotic

Endothelial cells produce reactive oxygen species (ROS) via NADPH oxidase complexes, which mediate signaling for proliferation, migration and cell death. We examined the roles of the Nox2‐ and Nox4‐containing NADPH oxidases in proliferation and survival of human microvascular endothelial cells (HMEC‐1). Superoxide and ROS production as well as Nox4 protein expression, but not Nox2 protein, were higher in proliferating than in quiescent HMEC‐1. Incubation of proliferating HMEC‐1 with diphenyleneiodinium, tiron (ROS scavenger), or Nox2‐ or Nox4‐specific siRNA, reduced ROS production and cell number. During proliferation, phosphorylation of ERK1/2 and Akt was increased. Inhibition of Nox4‐derived ROS production reduced both ERK1/2 and Akt phosphorylation, whereas inhibition of Nox2 only reduced the Akt phosphorylation. Furthermore, suppression of Nox2 but not Nox4 caused activation of caspase 3/7. In endothelial cells, Nox4‐derived ROS promote ERK1/2 and Akt signaling and proliferation, whereas Nox2 activity increases Akt and caspase 3/7 and prevents apoptosis. Isoform‐selective modulation of NADPH oxidase could be a useful therapeutic approach for either blocking or stimulating angiogenesis, as required for regenerative repair of ischemic or damaged tissues.

Antiangiogenic properties of fasudil, a potent Rho-Kinase inhibitor

Vascular endothelial growth factor (VEGF) plays a pivotal role in pathological angiogenesis. In this study, we addressed the therapeutic potential of fasudil, a potent Rho-kinase inhibitor, for VEGF-elicited angiogenesis and also for the intracellular signalings induced by VEGF. In vitro, the inhibitory effects of fasudil on the VEGF-dependent VEGF receptor 2 (VEFGR2 or KDR), extracellular signal-related kinase (ERK) 1/2, Akt and myosin light chain (MLC) phosphorylation, as well as on the migration and proliferation of bovine retinal microvascular endothelial cells (BRECs) were analyzed with Western blotting, [3H]-thymidine uptake, and modified Boyden chamber assay. VEGF-elicited in vivo angiogenesis was analyzed with a mouse corneal micropocket assay coembedded with or without fasudil. VEGF caused enhanced MLC phosphorylation of BRECs, which was almost completely attenuated by 10microM fasudil. VEGF-dependent phosphorylation of ERK1/2 and Akt were also partially but significantly attenuated by treatment with fasudil without affecting VEGFR2 (KDR) phosphorylation. Moreover, both VEGF-induced [3H]-thymidine uptake and the migration of BRECs were significantly inhibited in the presence of fasudil. Finally, VEGF-elicited angiogenesis in the corneal micropocket assay was potently attenuated by coembedding with fasudil (P < 0.01). These findings indicate that fasudil might have a therapeutic potential for ocular angiogenic diseases. The antiangiogenic effect of fasudil appears to be mediated through the blockade not only of Rho-kinase signaling but also of ERK and Akt signaling.

The Kringle 1 Domain of Hepatocyte Growth Factor Has Antiangiogenic and Antitumor Cell Effects on Hepatocellular Carcinoma

The kringle 1 domain of human hepatocyte growth factor (HGFK1) was previously shown to inhibit bovine aortic endothelial cell proliferation, suggesting that it might be an antiangiogenic molecule. Here, we evaluated the in vivo efficacy of a recombinant adenoassociated virus carrying HGFK1 (rAAV-HGFK1) for the treatment of hepatocellular carcinoma (HCC) in a rat orthotopic HCC model and explored its molecular mechanisms in vitro in both endothelial and tumor cells. We first showed that rAAV-HGFK1 treatment significantly prolonged the survival time of rats transplanted with tumor cells. Treatment with rAAV-HGFK1 inhibited tumor growth, decreased tumor microvessel density, and completely prevented intrahepatic, lung, and peritoneal metastasis in this in vivo model. In vitro, rAAV-HGFK1 exhibited both antiangiogenic and antitumor cell effects, inhibiting the proliferation of both murine microvascular endothelial cells (MEC) and tumor cells, and inducing apoptosis and G(0)-G(1) phase arrest in these cells. To our surprise, rAAV-HGFK1 did not act through the hepatocyte growth factor/hepatocyte growth factor receptor pathway. Instead, it worked mainly through epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling, with more minor contributions from vascular endothelial growth factor/vascular endothelial growth factor receptor and beta fibroblast growth factor (bFGF)/beta fibroblast growth factor receptor (bFGFR) signaling. In both MECs and tumor cells, rAAV-HGFK1 acted through two pathways downstream of EGFR, namely inhibition of extracellular signal-regulated kinase activation and stimulation of p38 mitogen-activated protein kinase/c-Jun-NH(2)-kinase activation. These results suggest for the first time that HGFK1 exerts both antiangiogenic and antitumor cell activities mainly through EGF/EGFR signaling, and may thus be considered as a novel therapeutic strategy for the treatment of HCC.

CALRETICULIN DOWNREGULATION IS ASSOCIATED WITH FGF-2-INDUCED ANGIOGENESIS THROUGH CALCINEURIN PATHWAY IN ISCHEMIC MYOCARDIUM

Fibroblast growth factor 2 (FGF-2) plays an integral role in therapeutic angiogenesis associated with myocardial infarct healing. Calcium (Ca(2+)) is one of the most universal important signaling molecules that affect cell proliferation and angiogenesis. Calreticulin (CRT), a 46-kd (Ca(2+)) -binding chaperone found mainly in the endoplasmic reticulum, plays an important role in regulating calcium homeostasis. The role of CRT in FGF-2-induced angiogenesis and its signaling pathways in ischemic myocardium are not clear. For this study, two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry were used to analyze CRT's differential expression in myocardial microvascular endothelial cells treated with or without FGF-2. Western blotting analysis was used to detect the expression of CRT and calcineurin (CaN) in sham-operated, FGF-2-, or saline intramyocardially injected myocardium. It is found that FGF-2 induced angiogenesis after sustained ischemia with downregulation of CRT expression and upregulation of CaN expression in myocardium. The CRT expression was negatively correlated to angiogenesis. Furthermore, overexpression of CRT or inhibition of CaN with cyclosporine A abolishes FGF-2-induced microvascular endothelial cells proliferation and CaN expression. The results indicate that intramyocardial administration of FGF-2 decreases myocardial CRT expression in parallel with myocardial angiogenesis in ischemic myocardium. The study further indicates that Ca(2+)/CaN signaling pathway may be involved in CRT-related angiogenesis.

Abstract 327: NADPH Oxidase-dependent Signaling Involved In Endothelial Cell Survival And Proliferation: Role Of Nox2 And Nox4

Objective: Endothelial cells (EC) produce a variety of reactive oxygen species (ROS) which act as mediators of signaling for proliferation, migration, differentiation and cell death. The major source of ROS in EC at rest is the NADPH oxidase enzyme complex. We aimed to determine the roles of the Nox2 and Nox4 catalytic subunits of NADPH oxidase in proliferation and survival of human microvascular endothelial cells (HMEC-1). Methods: Intracellular superoxide was measured by dihydroethidium fluorescence and lucigenin chemiluminescence, and total ROS by dichlorodihydrofluorescein-diacetate assay. Nox4 and Nox2 protein expression was measured by Western blot, and cell proliferation was quantitated by trypan blue exclusion and a tetrazolium-based MTS assay. EC survival was measured by caspase3/7 activity. Results: The expression of Nox4 (but not Nox2) protein, superoxide and total ROS, were all significantly higher (P &lt; 0.05, N = 4) under proliferative conditions (sparse culture or serum addition) than in quiescent (dense culture or serum-starved) cells. Suppression of NADPH oxidase-derived ROS with specific siRNA significantly reduced cell proliferation as shown below. Under proliferative conditions phosphorylation of ERK and Akt were increased. Suppression of Nox4-derived ROS production reduced the phosphorylation of both ERK and Akt, whereas suppression of Nox2 reduced only Akt phosphorylation. Further, suppression of Nox2, but not Nox4, significantly activated caspase3/7 (P &lt; 0.05, N = 4). Thus Nox4-derived ROS act to promote proliferation of EC via ERK and Akt signaling, whereas Nox2 prevents apoptotic signaling via Akt and caspase3/7. Conclusion: Nox isoform-derived ROS act via distinct signaling pathways to promote EC proliferation and survival. Selective modulation of Nox-type NADPH oxidase could be a useful approach for blocking angiogenesis or for therapeutic angiogenesis in chronic ischemia or tissue engineering.

713 POSTER Molecularly targeted immunochemotherapeutic formulation (SEVINA) composed of pegylated trispecific disulfide linked Fv (sdFv) targeting epitopes of EGFR, PTHrP and RANKL conjugated covalently with SATA to vinorelbine eradicated osteolytic metastases of multiple myeloma inhibiting proliferation of tumor associated and bone microvascular endothelial cells inducing ADCC, AMP and type I, II, III PCD

Highly promising preclinical data obtained in cultured cells and in nude mice bearing xenografts contrast with the rather modest clinical efficacy of Polo-like kinase 1 (Plk1) inhibitors. In the present study, we investigated if Plk1 might be a suitable target in hepatocellular carcinoma (HCC) and if a genetically engineered mouse tumor model that well reflects the tumor cell and micro-environmental features of naturally occurring cancers might be suitable to study anti-Plk1 therapy. Analysis of Plk1 expression in human HCC samples confirmed that HCC express much higher Plk1 levels than the adjacent normal liver tissue. Inhibition of Plk1 by an adenovirus encoding for a short hairpin RNA against Plk1 or by the small-molecule inhibitor BI 2536 reduced the viability of HCC cell lines and inhibited HCC xenograft progression in nude mice. Treatment of transforming growth factor (TGF) α/c-myc bitransgenic mice with BI 2536 during hepatocarcinogenesis reduced the number of dysplastic foci and of Ki-67-positive cells within the foci, indicating diminished tumorigenesis. In contrast, BI 2536 had no significant effect on HCC progression in the transgenic mouse HCC model as revealed by magnetic resonance imaging. Measurement of BI 2536 by mass spectrometry revealed considerably lower BI 2536 levels in HCC compared with the adjacent normal liver tissue. In conclusion, low intratumoral levels are a novel mechanism of resistance to the Plk1 inhibitor BI 2536. Plk1 inhibitors achieving sufficient intratumoral levels are highly promising in HCC treatment.

Nanostructured HA crystals up-regulate FGF-2 expression and activity in microvascular endothelium promoting angiogenesis

In mineralized tissue the process of angiogenesis is required for normal osteogenesis during bone repair and in reconstructive and substitutive surgery, for proper biomaterial/tissue integration. Nanotechnologies have been proposed to improve the compatibility of biomaterials for use in orthopaedic and reconstructive surgery (e.g., nanocrystals). The aim of this study was to determine the effect of nanostructured hydroxyapatite (HA) on angiogenesis. Microvascular endothelial cell survival, proliferation and migration, crucial events in the angiogenic process, were evaluated together with cytoskeleton and biochemical signalling markers. Induction of migration, metalloproteinase (MMP-2) and focal adhesion Kinase (FAK) activity documented the ability of HA nanocrystals to stimulate capillary endothelium toward an angiogenic phenotype. HA concentrations, ranging from 2 to 10 μg/ml, promoted endothelium survival and proliferation, preserved α vβ 3-integrin localization, stimulated β-actin reorganization and Akt phosphorylation (98% vs control). Immunoassays for key signalling pathways in angiogenesis (i.e., endothelial nitric oxide synthase (eNOS) and fibroblast growth factor-2 (FGF-2)) demonstrated that HA increased their expression. Moreover, quantitative RT-PCR and Western blotting analysis confirmed that HA nanocrystals exposure up-regulated FGF-2 mRNA by 6 fold and increased 18 kDa protein isoform by 40%. HA enhanced cell responsiveness to vascular endothelial growth factor (VEGF) in terms of NOS activity (1.5 fold over control), increasing the ability of microvascular endothelium to differentiate into capillary-like structures when grown in 3D fibrin gel. In conclusion our data document the proangiogenic properties of HA nanocrystals. This material stimulates endothelial cell functions and biochemical pathways to an extent similar to VEGF, and primes them to VEGF stimulation, leading to differentiation in pseudocapillary formations in 3D matrices.

Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988–2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

Comprehensive investigation of disease‐specific short peptides in sera from patients with systemic sclerosis: Complement C3f‐des‐arginine, detected predominantly in systemic sclerosis sera, enhances proliferation of vascular endothelial cells

To identify pathogenic and/or disease-specific short peptides in sera from patients with systemic sclerosis (SSc). Serum samples from 40 patients with SSc, 30 patients with systemic lupus erythematosus, 21 patients with rheumatoid arthritis, 30 patients with osteoarthritis, and 26 healthy donors were tested. Short peptides with molecular weights of smaller than approximately 3 kd, purified from the sera by magnetic bead-based hydrophobic interaction chromatography 18, were detected and their amino acid sequences determined using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Effects of the identified peptides on fibroblasts and microvascular endothelial cells were tested using synthesized peptides and sera containing the peptides. A group of peptides with mass/charge (m/z) values of 1,865, 1,778, 1,691, 1,563, and 1,450 were detected predominantly in the SSc sera. These peptides were identified as family members of complement C3f-des-arginine (DRC3f) derived from C3b. The level of DRC3f (m/z 1,865) was related to vascular involvement in SSc and to SSc disease activity. The synthesized peptides of DRC3f and C3f, as well as the filtrated sera containing DRC3f, enhanced proliferation of microvascular endothelial cells, but not fibroblasts. Both DRC3f and C3f increased production of transforming growth factor beta1 by dermal microvascular endothelial cells. This comprehensive peptidomics analysis revealed the predominance of DRC3f in the sera of patients with SSc. Investigation of DRC3f may be a useful tool for the diagnosis and evaluation of disease activity in SSc. Moreover, its demonstrated effects on endothelial cells suggest a potential role for DRC3f in the pathophysiologic mechanisms of SSc.

Interaction of α9β1 Integrin With Thrombospondin-1 Promotes Angiogenesis

Thrombospondin-1 is a multifunctional protein interacting with several cell surface receptors including integrins. We found that it is a ligand for alpha9beta1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with alpha9beta1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing alpha9beta1 integrin activated signaling proteins such as Erk1/2 and paxillin. Blocking of this integrin by monoclonal antibody and the met-leu-asp-disintegrin inhibited dermal human microvascular endothelial cell proliferation and NoC1-induced migration of these cells. Immunohistochemical studies revealed that alpha9beta1 is expressed on microvascular endothelium in several organs including skin, lung, heart and brain. NoC1 induced neovascularization in an experimental quail chorioallantoic membrane system and Matrigel plug formation assay in mice. This proangiogenic activity of NoC1 in vivo was inhibited by alpha9beta1 inhibitors. In summary, our results revealed that alpha9beta1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.

Stimulation of cerebral and dermal microvascular endothelial cell proliferation by AT1 receptor antagonist losartan

Aim: We have shown that cerebral angiogenesis is stimulated by chronic treatment of rats with the angiotensin II (ANG II) type 1 receptor (AT1R) antagonist, losartan (LOS). Because the trophic actions of ANG II via AT1R are well known, the finding that an AT1R antagonist increases angiogenesis is paradoxical. The aim is to determine whether cerebral (CMVEC) and dermal (DMVEC) microvascular endothelial cells differ in their growth responses to LOS treatment. Methods: Primary human CMVEC and DMVEC, passage 3–8, were seeded into 96-well plates (5000 cells/well) and allowed to adhere for 24 hrs. Cells were exposed to serum-free media (SFM) alone and in the presence of LOS (10 μM), ANG II (1 μM) or both for 48 hours. Cell density was quantified by a fluorescent dye proliferation assay at 480/520 nm. Results: Density of CMVEC increased with either ANG II stimulation (27.5%) or treatment with LOS, with (33.7%) or without ANG II (27.1%) compared to SFM alone. A similar but less robust pattern was seen with DMVEC with ANG II (8.9%), LOS + ANG II (15.2%) and LOS alone (19.0%). Conclusion: This suggests that CMVEC and DMVEC have similar proliferative responses so they do not appear to be tissue-specific. However, LOS alone has a similar effect to treatment with both the agonist and antagonist suggesting that LOS directly stimulates cell proliferation and that the effect is not due to receptor blockade per se. Supported by NIH HL29587 and HV28182