Human Umbilical Vein-derived Endothelial Cells Research Articles

Spatial control of self-organizing vascular networks with programmable aptamer-tethered growth factor photopatterning.

Given the dynamic nature of engineered vascular networks within biofabricated tissue analogues, it is instrumental to have control over the constantly evolving biochemical cues within synthetic matrices throughout tissue remodeling. Incorporation of pro-angiogenic vascular endothelial growth factor (VEGF165) specific aptamers into cell-instructive polymer networks is shown to be pivotal for spatiotemporally controlling the local bioactivity of VEGF that selectively elicit specific cell responses. To harness this effect and quantitatively unravel its spatial resolution, herein, bicomponent micropatterns consisting of VEGF165 specific aptamer-functionalized gelatin methacryloyl (GelMA) (aptamer regions) overlaid with pristine GelMA regions using visible-light photoinitiators (Ru/SPS) were fabricated via two-step photopatterning approach. For the 3D co-culture study, human umbilical vein-derived endothelial cells and mesenchymal stromal cells were used as model cell types. Bicomponent micropatterns with spatially defined spacings (300/500/800​μm) displayed high aptamer retention, aptamer-fluorescent complementary sequence (CSF) molecular recognition and VEGF sequestration localized within patterned aptamer regions. Stiffness gradient at the interface of aptamer and GelMA regions was observed with high modulus in aptamer region followed by low stiffness GelMA regions. Leveraging aptamer-tethered VEGF's dynamic affinity interactions with CS that upon hybridization facilitates triggered VEGF release, co-culture studies revealed unique characteristics of aptamer-tethered VEGF to form spatially defined luminal vascular networks covered with filopodia-like structures in vitro (spatial control) and highlights their ability to control network properties including orientation over time using CS as an external trigger (temporal control). Moreover, the comparison of single and double exposed regions within micropatterns revealed differences in cell behavior among both regions. Specifically, the localized aptamer-tethered VEGF within single exposed aptamer regions exhibited higher cellular alignment within the micropatterns till d5 of culture. Taken together, this study highlights the potential of photopatterned aptamer-tethered VEGF to spatiotemporally regulate vascular morphogenesis as a tool for controlling vascular remodeling in situ.

Spatiotemporally controlled, aptamers-mediated growth factor release locally manipulates microvasculature formation within engineered tissues

Spatiotemporally controlled growth factor (GF) delivery is crucial for achieving functional vasculature within engineered tissues. However, conventional GF delivery systems show inability to recapitulate the dynamic and heterogeneous nature of developing tissue's biochemical microenvironment. Herein, an aptamer-based programmable GF delivery platform is described that harnesses dynamic affinity interactions for facilitating spatiotemporal control over vascular endothelial GF (VEGF165) bioavailability within gelatin methacryloyl matrices. The platform showcases localized VEGF165 sequestration from the culture medium (offering spatial-control) and leverages aptamer-complementary sequence (CS) hybridization for triggering VEGF165 release (offering temporal-control), without non-specific leakage. Furthermore, extensive 3D co-culture studies (human umbilical vein-derived endothelial cells & mesenchymal stromal cells), in bi-phasic hydrogel systems revealed its fundamentally novel capability to selectively guide cell responses and manipulate lumen-like microvascular networks via spatiotemporally controlling VEGF165 bioavailability within 3D microenvironment. This platform utilizes CS as an external biochemical trigger for guiding vascular morphogenesis which is suitable for creating dynamically controlled engineered tissues.

Construction of transplantable artificial vascular tissue based on adipose tissue-derived mesenchymal stromal cells by a cell coating and cryopreservation technique

Prevascularized artificial three-dimensional (3D) tissues are effective biomaterials for regenerative medicine. We have previously established a scaffold-free 3D artificial vascular tissue from normal human dermal fibroblasts (NHDFs) and umbilical vein-derived endothelial cells (HUVECs) by layer-by-layer cell coating technique. In this study, we constructed an artificial vascular tissue constructed by human adipose tissue-derived stromal cells (hASCs) and HUVECs (ASCVT) by a modified technique with cryopreservation. ASCVT showed a higher thickness with more dense vascular networks than the 3D tissue based on NHDFs. Correspondingly, 3D-cultured ASCs showed higher expression of several angiogenesis-related factors, including vascular endothelial growth factor-A and hepatic growth factor, compared to that of NHDFs. Moreover, perivascular cells in ASCVT were detected by pericyte markers, suggesting the differentiation of hASCs into pericyte-like cells. Subcutaneous transplantation of ASCVTs to nude mice resulted in an engraftment with anastomosis of host’s vascular structures at 2 weeks after operation. In the engrafted tissue, the vascular network was surrounded by mural-like structure-forming hASCs, in which some parts developed to form vein-like structures at 4 weeks, suggesting the generation of functional vessel networks. These results demonstrated that cryopreserved human cells, including hASCs, could be used directly to construct the artificial transplantable tissue for regenerative medicine.

Abstract 5240: Effective inhibition of TMPRSS2-ERG positive prostate cancer cells by a new formula of ERGi-USU-6

Abstract Background: Prostatic adenocarcinoma is among the leading cause of cancer-related deaths among men in the United States. The Erythroblast Transformation-Specific-Related Gene, ERG is dormant in normal prostate epithelium. Due to gene fusions, the ERG oncogene is frequently activated by androgenic signals in prostate cancer. ERG disrupts the normal differentiation, promotes epithelial-mesenchymal transition, migratory and invasive properties of cancer cells. Approximately 35% of metastatic castration-resistant prostate cancers harbor ERG oncogene. Due to the failure of androgen axis directed therapies, there is an urgent need to develop inhibitors to targeting prostate cancer-causing genes, such as ERG. We have identified a potent small-molecule inhibitor ERGi-USU that is remarkably selective for inhibiting the growth of ERG positive cancer cells through direct binding to the RIOK2 atypical kinase, a putative upstream regulator of ERG. We completed a structure-activity relationship (SAR) study and compound development resulting in the potent derivative, ERGi-USU-6. Our current objective is to improve the therapeutic properties of ERGi-USU-6, by new salt formulations. Methods: Evaluation of the five selected salt formulations of ERGi-USU-6 were performed by assessing the growth of ERG positive prostate cancer cell line (VCaP) and by monitoring ERG and RIOK2 protein levels. Selectivity was assessed by monitoring the growth, endogenous ERG and RIOK2 levels in normal ERG positive human umbilical vein derived endothelial cells (HUVEC). The IC50 values for cell growth, compared to the parental compound were calculated in a 12-step dilution range performed in triplicates and independently repeated three times. Cell growth was measured by quantitative Cell Glow assay monitoring viable cells using Perkin Elmer Envision assay instrument and protein levels were quantified by measuring bioluminescence in IBright instrument in a wide dynamic range. Results: One new salt formula with improved activity were identified, demonstrating improved cell growth (IC50=89nM vs. parental IC50=139), ERG protein and RIOK2 protein inhibition. None of the salt formulas of ERGi-USU-6 showed any effect on the primary cultures of the ERG positive normal endothelial cells (HUVEC) in the effective concentration range. Conclusion: The first evaluation of salt formulas of ERGi-USU-6 may open the possibilities for preclinical assessments of this remarkably cancer-selective compound. Citation Format: Binil Eldhose, Charles P. Xavier, Mallesh Pandrala, Sanjay V. Malhotra, Albert Dobi. Effective inhibition of TMPRSS2-ERG positive prostate cancer cells by a new formula of ERGi-USU-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5240.

In Vitro Angiogenic Properties of Plasmid DNA Encoding SDF-1α and VEGF165 Genes.

The stromal-derived factor-1 alpha (SDF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and exert a significant trophic function. SDF-1α is a chemoattractant for endothelial progenitor cells derived from bone marrow and promotes new blood vessel formation. VEGF regulates all types of vascular growth, stimulates angiogenesis, and is involved in the induction of lymphangiogenesis. The possibility of using these growth factors for regenerative medicine is currently under investigation. The angiogenic potential of a pBud-SDF-1α-VEGF165 bicistronic plasmid construct which simultaneously encodes VEGF165 and SDF-1α genes cDNA was evaluated in this study. The conditioned medium collected from HEK293T cells transfected with the pBud-SDF-1α-VEGF165 plasmid was shown to stimulate the formation of capillary-like structures by human umbilical vein-derived endothelial cells (HUVEC) on Matrigel and to increase the proliferative activity of these cells in vitro. Thus, the pBud-SDF-1α-VEGF165 plasmid exhibits angiogenic properties in cell cultures in vitro. As interest in the development of non-viral techniques for regenerative medicine increases, this plasmid which simultaneously expresses VEGF165 and SDF-1α may provide a platform for advanced methods of stimulating therapeutic angiogenesis.

Omega-3 polyunsaturated fatty acids promote angiogenesis in placenta derived mesenchymal stromal cells

Enhancement of angiogenesis is solicited in wound repair and regeneration. Mesenchymal stromal cells derived from the placenta (P-MSCs) have an inherent angiogenic potential. Polyunsaturated fatty acids (PUFAs) in turn, specifically the omega-3 (N-3) are essential for growth and development. They are also recommended as dietary supplements during pregnancy. We therefore hypothesized that addition of N-3 PUFAs in P-MSC culture media may enhance their angiogenic potential. Hence, we treated P-MSCs with omega-3 (N-3) fatty acids –Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) at different concentrations and tested their angiogenic potential. We saw an upregulation of both bFGF and VEGFA. We also found enhanced in vitro tube formation ability of P-MSCs treated with DHA: EPA. We then looked at the influence of the conditioned medium (CM) collected from P-MSCs exposed to DHA: EPA on the key effector cells –HUVECs (Human Umbilical Vein derived endothelial cells and their functionality was further confirmed on chick yolk sac membrane. We found that the CM of P-MSCs exposed to DHA: EPA could enhance angiogenesis in both cases. These result were finally validated in an in vivo matrigel plug assay which revealed enhanced migration and vessel formation in CM treated with DHA: EPA. Our data thus reveals for the first time that supplementation with lower concentration of PUFA enhances the angiogenic potential of P-MSCs making them suitable for chronic wound healing applications.

Towards a Biohybrid Lung Assist Device: N-Acetylcysteine Reduces Oxygen Toxicity and Changes Endothelial Cells' Morphology.

The development of an endothelialized membrane oxygenator requires solution strategies combining the knowledge of oxygenators with endothelial cells' biology. Since it is well known that exposing cells towards pure oxygen causes oxidative stress, this aspect has to be taken into account in the development of a biohybrid oxygenator system. N-Acetylcysteine (NAC) is known for its antioxidant properties in cells. We tested its applicability for the development of an endothelialized oxygenator model. Cultivating human umbilical vein derived endothelial cells (HUVEC) up to 6days with increasing concentrations of NAC from 1 to 30mM revealed NAC toxicity at concentrations from 20mM. Cell density clearly decreased after radical oxygen species exposure in non-NAC pretreated cells compared to 20mM NAC precultured HUVEC after 3 and 6days. Also the survival rate after ROS treatment could be restored by incubation with NAC from 15 to 25mM for all time points. NAC treated cells changed their morphology from typical endothelial cells' cobblestone pattern to a fusiform, elongated configuration. Transformed cells were still positive for typical endothelial cell markers. Our present results show the potential of NAC for the protection of an endothelial cell layer in an endothelialized membrane oxygenator due to its antioxidative properties. Moreover, NAC induces a morphological change in HUVEC similar to dynamic cultivation procedures.

A SEMA3E mutant resistant to cleavage by furins (UNCL-SEMA3E) inhibits choroidal neovascularization

Abnormal subretinal choroidal neovascularization (CNV) is a major cause of blindness in exudative age-related macular degeneration (AMD). Current anti-angiogenic treatments by VEGF sequestering agents have been successful, but a significant proportion of patients do not respond well to these treatments, and the response of others diminishes over time, suggesting that additional anti-angiogenic agents that function by separate mechanisms may be of use to such patients. We have previously found that a point mutated form of semaphorin-3E resistant to cleavage by furin like pro-protein convertases (UNCL-Sema3E) displays potent anti-angiogenic properties. We therefore determined if UNCL-Sema3E has potential as an inhibitor of CNV formation. We chose to study UNCL-Sema3E rather than wild type sema3E because unlike full length sema3E, the major p61-Sema3E peptide that is produced by cleavage of sema3E with furin like pro-protein convertases activates signal transduction mediated by the ErbB2 receptor and can promote tumor metastasis in addition to its anti-angiogenic activity. UNCL-Sema3E inhibited efficiently vascular endothelial growth factor-A (VEGF), platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) signaling in human umbilical vein derived endothelial cells (HUVEC) and to a lesser extent hepatocyte growth factor (HGF) signal transduction. CNV that was induced in the eyes of C57 black mice by laser photocoagulation was inhibited by 65% (P < 0.01) following a single bolus intra-vitreal injection of 5 μg UNCL-Sema3E. This inhibitory effect was similar to the inhibition produced by a single bolus intra-vitreal injection of 5 μg aflibercept. A similar inhibition of CNV was observed following the injection of UNCL-Sema3E into the eyes of Long-Evans rats. However, a higher dose of UNCL-Sema3E (125 μg), partially due to the larger volume of the vitreous cavity of rats, was required to achieve maximal inhibition of CNV. Injection of UNCL-Sema3E into eyes of healthy mice did not have any adverse effect on retinal function as assessed by optic kinetic reflex (OKR) or by electroretinogram (ERG) assays nor did UNCL-Sema3E injection affect the structure of the retina as determined using histology. To conclude, our results suggest that UNCL-Sema3E may be useful for the treatment of exudative AMD, which does not respond well to conventional anti-VEGF therapy.

Angiopoietin‐2 mediates thrombin‐induced monocyte adhesion and endothelial permeability

Essentials Mechanism of thrombin-induced inflammation is not fully understood. Thrombin induced monocyte adhesion and barrier loss require Angiopoietin-2 (Ang-2). Ang-2 mediates vessel leakage and monocyte adhesion through SHP-2/p38MAPK pathway. Calcium dependent SHP2/p38MAPK activation regulates Ang-2 expression through a feedback loop. Background Thrombin imparts an inflammatory phenotype to the endothelium by promoting increased monocyte adhesion and vascular permeability. However, the molecular players that govern these events are incompletely understood. Objective The aim of this study was to determine whether Angiopoietin-2 (Ang-2) has a role, if any, in regulating inflammatory signals initiated by thrombin. Methods Assessment of vascular leakage by Miles assay was performed by intra-dermal injection on the foot paw. Surface levels of intercellular adhesion molecule-1 (ICAM-1) were determined by flow cytometry. Overexpression, knockdown and phosphorylation of proteins were determined by Western blotting. Results In time-course experiments, thrombin-stimulated Ang-2 up-regulation, peaked prior to the expression of adhesion molecule ICAM-1 in human umbilical vein-derived endothelial cells (HUVECs). Knockdown of Ang-2 blocked both thrombin-induced monocyte adhesion and ICAM-1 expression. In addition, Ang-2(-/-) mice displayed defective vascular leakage when treated with thrombin. Introducing Ang-2 protein in Ang-2(-/-) mice failed to recover a wild-type phenotype. Mechanistically, Ang-2 appears to regulate the thrombin-activated calcium spike that is required for tyrosine phosphatase SHP2 and p38 MAPK activation. Further, down-regulation of SHP2 attenuated both thrombin-induced Ang-2 expression and monocyte adhesion. Down-regulation of the adaptor protein Gab1, a co-activator of SHP2, as well as overexpression of the Gab1 mutant incapable of interacting with SHP2 (YFGab1), inhibited thrombin-mediated effects, including downstream activation of p38 MAPK, which in turn was required for Ang-2 expression. Conclusions The data establish an essential role of the Gab1/SHP2/p38MAPK signaling pathway and Ang-2 in regulating thrombin-induced monocyte adhesion and vascular leakage.

Influence of cell‐internalization on relaxometric, optical and compositional properties of targeted paramagnetic quantum dot micelles

Quantum dot micelles (pQDs) with a paramagnetic coating are promising nanoparticles for bimodal molecular imaging. Their bright fluorescence allows for optical detection, while their Gd payload enables visualization with contrast-enhanced MRI. A popular approach in molecular MRI is the targeting of abundantly expressed cell surface receptors. Ligand-receptor binding often results in cell internalization of the targeted contrast agent. The interpretation of molecular imaging with pQDs therefore requires knowledge about the consequences of cellular internalization for their relaxometric, optical and compositional properties. To study these, Cd-containing core-shell-shell QDs coated with a monolayer of lipids, of which 50 mol% was a Gd-containing lipid, were incubated with human umbilical vein-derived endothelial cells (HUVECs) for up to 24 h. α(ν) β(3)-integrin targeted (RGD) and non-targeted (NT) pQDs were compared. pQDs uptake was monitored by fluorescence microscopy, FACS, ICP-MS, relaxometry and MRI. Cell-associated pQDs displayed longitudinal relaxation rates and fluorescent intensities which were linear with the cell-associated Gd and Cd concentrations, implying that the Gd and Cd uptake by HUVECs can be quantified using relaxometric and optical measurements, respectively. However, the Gd-to-Cd molar ratio in pellets of pQD-incubated cells was consistently higher than the Gd-to-Cd molar ratio of the pQDs as prepared. It is proposed that this increase in Gd-to-Cd molar ratio was due to non-specific lipid-transfer between the pQDs and the cellular membranes. These findings show that, in the case of contrast agents that are formed by non-covalent interactions, experimental procedures are needed with which representative components of the probes can be monitored.

Selective activation of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3 in human endothelial cells infected with Rickettsia rickettsii

A majority of typical pathologic sequelae of rickettsial diseases, such as altered barrier function of endothelial cells leading to compromised vascular permeability, manifest as non-cardiogenic pulmonary oedema ⁄ acute respiratory distress syndrome and parenchymal cerebral oedema, which can be attributed to disseminated infection and damage of the vascular endothelium [1]. Endothelial cells infected in vitro with R. rickettsii or R. conorii undergo profound phenotypic modifications to an ‘activated’ state due to induction of a number of immediate early responsive prothrombotic and proadhesive genes [2]. Ample published evidence also indicates an important role for interferons (IFNs) in host response and clearance of pathogenic Rickettsia species [3]. In general, both type I (a, b) and type II (c) IFNs modulate the innate immune response through Janus kinase (JAK)-STAT (Signal transducer and activator of transcription) mediated gene regulation. JAK-STAT pathway transmits signals originating from extracellular ligands and those from within the cell directly to the nucleus to control various gene promoters. There are four known Janus kinases (JAK1-3 and TYK2) and seven STAT proteins (STAT1-4, 5a, 5b, and 6). To better understand the immune response of vascular endothelium to infection with spotted fever rickettsiae, we have initiated efforts to investigate the contributions of the JAK-STAT signalling pathway to the pathophysiology of human spotted fever group (SFG) rickettsioses. Standard laboratory protocols established and described earlier were used for in vitro infection of cultured human umbilical vein-derived endothelial cells (HUVECs) [4]. Briefly, cells were incubated in antibiotic-free culture medium with approximately 6 · 10 pfu of R. rickettsii organisms for every cm of culture area. Indirect immunofluorescent staining and citrate synthase (gltA)-based quantitative PCR confirmed that this experimental protocol consistently resulted in infection of ‡80% of cells with three to five rickettsiae at 6 h [5]. Because phosphorylation of specific target residues triggers subsequent dimerisation and nuclear translocation, activation of different STAT proteins was determined by denaturing polyacrylamide gel electrophoresis of total protein lysates from R. rickettsii-infected HUVECs followed by Western blot analysis using phosphorylation state-specific antibodies. As expected, uninfected endothelial cells were found to have either very low or negligible basal phosphorylation levels of all investigated STAT proteins. In response to R. rickettsii infection, however, the phosphorylation of STAT3 and STAT1 was clearly induced (Fig. 1), while that of STAT2, STAT5 and STAT6 remained unaltered at all times (not shown). Interestingly, there were noticeable differences in the kinetics of activation of STAT1 and STAT3 in response to infection. The levels of p-STAT3 in infected cells were two to three times higher than corresponding controls at early times of 1.5 and 3 h and then declined to basal levels at 8 h post-infection (Fig. 1a). In contrast, the levels of p-STAT1 did not display significant changes early during the infection and started to increase at about 6 h post-infection with as much as four-fold and eight-fold induction at 12 h and 24 h post-infection, respectively (Fig. 1b). These results indicate that activation of only selective STAT proteins occurs in response to R. rickettsii infection and highlight the unique differences in kinetics of their activation. Corresponding author and reprint requests: Sanjeev K. Sahni, PhD, Department of Microbiology and Immunology, Room 211132, PO Box 672, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA E-mail: sanjeev_sahni@urmc.rochester.edu

Thalidomide and Thalidomide Transformed by pH-Dependent Hydrolysis or by Liver Enzyme Treatment Does Not Impede the Proliferation of Endothelial Cells

Angiogenesis is essential for tumor growth. In the treatment of tumors with thalidomide, a rationale for its use is that it inhibits angiogenesis. To form new blood vessels, endothelial cells must proliferate. The authors were interested in describing some of the circumstances by which thalidomide may influence proliferation of endothelial cells. Human umbilical vein derived endothelial cells (HUVECs) were exposed to 4.0 or 100 μg/ml of thalidomide in dimethylsulfoxide or to 4 μg/ml of thalidomide prepared an aqueous solutions. Proliferation was determined by measuring incorporation of 3H-thymidine. Regardless of the solvent used to dissolve thalidomide, the HUVECs treated with thalidomide were not inhibited in their ability to incorporate 3H-thymidine.

Establishment of a human in vitro model of the outer blood–retinal barrier

The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age-related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE-19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein-derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE-19. Both human umbilical vein-derived endothelial and ARPE-19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein-derived endothelial cells in the trilayer became fenestrated when co-cultured with the ARPE-19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE-19 cells. These cells also revealed VE-cadherin and ZO-1 at cell-cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO-1, were localized to cell-cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein-conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten-fold reduction) whilst transfer of a 4 kDa FITC-conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional-like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE-19 and human umbilical vein-derived endothelial cells can be co-cultured on the amniotic membrane and that the resultant cross-talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer-retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD.

Semaphorin-3A and Semaphorin-3F Work Together to Repel Endothelial Cells and to Inhibit Their Survival by Induction of Apoptosis

Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF(165)) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF(165) do not inhibit VEGF(165)-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF(121), a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF(165) activity does not depend on competition with VEGF(165) for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins.

Identification of a Neutralizing scFv Binding to Human Vascular Endothelial Growth Factor 165 (VEGF165) Using a Phage Display Antibody Library

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF121, 145, 165, 183, 189, 206). VEGF is essential for tumor angiogenesis, and several studies have correlated elevated VEGF levels with tumor stage, metastases, and progression. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-VEGF165. After four rounds of panning against VEGF165, 40 out of 90 phage clones displayed VEGF165-binding activity. One of the positive clones, designated B8, bound to VEGF165 with relatively high affinity and neutralized VEGF165 bioactivity in vitro. The B8 clone was expressed in the soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The purified scFv recognized VEGF165 with the K(D) of 1.80 x 10(-8) M without cross-reaction to VEGF121. In addition to binding, the purified scFv could does-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cells proliferation. Together with its fully human mature, B8 scFv may have therapeutic implications in therapy of angiogenesis-dependent diseases.

The Streptococcal Lipoprotein Rotamase A (SlrA) Is a Functional Peptidyl-prolyl Isomerase Involved in Pneumococcal Colonization

Streptococcus pneumoniae expresses two surface-exposed lipoproteins, PpmA and SlrA, which share homology with distinct families of peptidyl-prolyl isomerases (PPIases). In this study, we demonstrated for the first time that the lipoprotein cyclophilin, SlrA, can catalyze the cis-trans isomerization of proline containing tetrapeptides and that SlrA contributes to pneumococcal colonization. The substrate specificity of SlrA is typical for prokaryotic and eukaryotic cyclophilins, with Suc-Ala-Ala-Pro-Phe-p-nitroanilide (pNA) being the most rapidly catalyzed substrate. In a mouse pneumonia model the slrA knock-out D39DeltaslrA did not cause significant differences in the survival times of mice compared with the isogenic wild-type strain. In contrast, a detailed analysis of bacterial outgrowth over time in the nasopharynx, airways, lungs, blood, and spleen showed a rapid elimination of slrA mutants from the upper airways but did not reveal significant differences in the lungs, blood, and spleen. These results suggested that SlrA is involved in colonization but does not contribute significantly to invasive pneumococcal disease. In cell culture infection experiments, the absence of SlrA impaired adherence to pneumococcal disease-specific epithelial and endothelial non-professional cell lines. Adherence of the slrA mutant could not be restored by exogenously added SlrA. Strikingly, deficiency in SlrA did not reduce binding activity to host target proteins, but resulted in enhanced uptake by professional phagocytes. In conclusion, SlrA is a functional, cyclophilin-type PPIase and contributes to pneumococcal virulence in the first stage of infection, namely, colonization of the upper airways, most likely by modulating the biological function of important virulence proteins.

PavA ofStreptococcus pneumoniaeModulates Adherence, Invasion, and Meningeal Inflammation

Pneumococcal adherence and virulence factor A (PavA) is displayed to the cell outer surface of Streptococcus pneumoniae and mediates pneumococcal binding to immobilized fibronectin. PavA, which lacks a typical gram-positive signal sequence and cell surface anchorage motif, is essential for pneumococcal virulence in a mouse infection model of septicemia. In this report the impact of PavA on pneumococcal adhesion to and invasion of eukaryotic cells and on experimental pneumococcal meningitis was investigated. In the experimental mouse meningitis model, the virulence of the pavA knockout mutant of S. pneumoniae D39, which did not show alterations of subcellular structures as indicated by electron microscopic studies, was strongly decreased. Pneumococcal strains deficient in PavA showed substantially reduced adherence to and internalization of epithelial cell lines A549 and HEp-2. Similar results were obtained with human brain-derived microvascular endothelial cells and human umbilical vein-derived endothelial cells. Attachment and internalization of pneumococci were not significantly affected by preincubation or cocultivations of pneumococci with anti-PavA antisera. Pneumococcal adherence was also not significantly affected by the addition of PavA protein. Complementation of the pavA knockout strain with exogenously added PavA polypeptide did not restore adherence of the mutant. These data suggest that PavA affects pneumococcal colonization by modulating expression or function of important virulence determinants of S. pneumoniae.

Platelet Factor 4 and Interleukin-8 CXC Chemokine Heterodimer Formation Modulates Function at the Quaternary Structural Level

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.

Immunofluorescent detection of activation of initiator caspases-8 and -9 during pharmacologically induced apoptosis of cultured HeLa and endothelial cells.

Caspases, a group of cysteine-activated aspartate-directed proteases, play an integral role in the execution of programmed cell death or apoptosis. In the cellular caspase cascade, the processing of native proenzymes into activated forms of downstream, effector caspases is dependent on the activation of initiator caspases-8 and -9. We describe a staining procedure for immunofluorescence-based analysis of activation of caspase-8 and -9 during pharmacologically induced apoptosis in primary cultures of human umbilical vein-derived endothelial cells and in an established line of HeLa cells. Using cleavage site-directed antibodies, specific intracellular detection for cleaved fragments of caspase-8 and -9 was accomplished during apoptosis induced by staurosporine and etoposide. The population of cells displaying morphological signs of apoptosis, evidence for DNA strand breaks by TUNEL analysis, and positive staining for active forms of caspase-8 and caspase-9 increased with the duration of treatment, suggesting activation of initiator caspases in correlation with the onset and progression of apoptosis. The application of immunocytochemical staining procedures for quick and specific in situ detection may effectively aid the identification of participating upstream caspases and elucidation of complex apoptosis signaling mechanisms.

Fibronectin mediates Opc-dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells.

A central step in the pathogenesis of bacterial meningitis caused by Neisseria meningitidis (the meningococcus) is the interaction of the bacteria with cells of the blood-brain barrier. In the present study, we analysed the invasive potential of two strains representing hypervirulent meningococcal lineages of the ET-5 and ET-37 complex in human brain-derived endothelial cells (HBEMCs). In contrast to previous observations made with epithelial cells and human umbilical vein-derived endothelial cells (HUVECs), significant internalization of encapsulated meningococci by HBMECs was observed. However, this uptake was found only for the ET-5 complex isolate MC 58, and not for an ET-37 complex strain. Furthermore, the uptake of meningococci by HBMECs depended on the presence of human serum, whereas serum of bovine origin did not promote the internalization of meningococci in HBMECs. By mutagenesis experiments, we demonstrate that internalization depended on the expression of the opc gene, which is present in meningococci of the ET-5 complex, but absent in ET-37 complex meningococci. Chromatographic separation of human serum proteins revealed fibronectin as the uptake-promoting serum factor, which binds to HBMECs via alpha 5 beta 1 integrin receptors. These data provide evidence for unique molecular mechanisms of the interaction of meningococci with endothelial cells of the blood-brain barrier and contribute to our understanding of the pathogenesis of meningitis caused by meningococci of different clonal lineages.