Human Umbilical Vein Endothelial Cells Attachment Research Articles

Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells

BackgroundEnhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. MethodsThe most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. ResultsOptimal concentration of each protein for surface coating was 50ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. ConclusionsSurfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device.

Sphingosine 1‐phosphate Potentiated Endothelial Cell Attachment on De‐cellularized Human Umbilical Vein as a Scaffold for Vascular Tissue Engineering

Cardiovascular disease (CVD), disorders of the heart and blood vessels, is the leading cause of mortality and morbidity. Arterial bypass graft is a primary therapy for the CVD patients. However, choosing a proper material for arterial bypass grafting is still a critical issue. Human umbilical vein (HUV) is a natural vessel and has been suggested as a suitable scaffold for vascular tissue engineering. In this study, we de‐cellularized HUV by using sodium dodecyl sulfate (SDS) and further incubated with medium contains 12% FBS to remove the residual nucleic acid. After the de‐cellularization process, HUV was examined by histological analysis as well as DNA quantification to conform the elimination of residual cellular composition. Human umbilical vein endothelial cells (HUVECs) were then seeded back on de‐cellularized HUV with the treatment of 1uM sphingosine 1‐phosphate (S1P). S1P is a lysophospholipid (LPL) which binds to G protein‐coupled receptors (GPCRs) and enhance endothelial cell proliferation, migration, and adhesion. Our results showed that S1P enhances HUVECs attachment on de‐cellularized HUV and reduce the time consuming for vessel grafts. The finding potentially provides a new strategy to develop vascular tissues.

A novel anti-platelet peptide (Z4A5) potential for glycoprotein IIb/IIIa inhibits platelet aggregation

IntroductionZ4A5 is a novel peptide that inhibits platelet aggregation and formation of platelet thrombi, but the mechanism of its anti-platelet effects remains unknown. This study explores the anti-platelet effect and mechanism of Z4A5. MethodsWe investigated the anti-platelet activity of Z4A5 on platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin (TH) in human platelet-rich plasma (PRP). Fibrinogen and PAC-1 binding to glycoproteinIIb/IIIa (GPIIb/IIIa) were measured by flow cytometry. In addition, we investigated the integrin specificity of Z4A5 in attachment and detachment assays using human umbilical vein endothelial cells (HUVEC) and assessed the relative cell number using the MTT assay. ResultsIn vitro, Z4A5 inhibited ADP-, AA- and TH-induced human platelet aggregation with IC50 values of 0.46±0.05μM (n=10), 0.23±0.05μM (n=10) and 0.21±0.02μM (n=10), respectively. Z4A5 inhibited fibrinogen, and PAC-1 bound to platelet GPIIb/IIIa with IC50 values of 0.48±0.07μM (n=8) and 0.63±0.12μM (n=6), respectively. Z4A5 failed to inhibit αVβ3 integrin-mediated HUVEC attachment to vitronectin and did not cause any significant detachment of HUVEC monolayer when compared with the controls. ConclusionsZ4A5 is a potential anti-platelet drug that inhibits fibrinogen binding to GPIIb/IIIa, but does not affect the structurally similar integrin αVβ3.

Viability, proliferation and adhesion of smooth muscle cells and human umbilical vein endothelial cells on electrospun polymer scaffolds

A major clinical problem of high relevance in the cardiovascular field is late stent thrombosis after implantation of drug eluting stents (DES). Clinical widely used DES currently utilize durable polymer coatings, which can induce persistent arterial wall inflammation and delayed vascular healing resulting in an impaired endothelialization. In this study we explored the interaction of smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) with electrospun scaffolds prepared from resorbable polyetheresterurethane (PDC) and poly(p-dioxanone) (PPDO), as well as polyetherimide (PEI), which can be surface modified, in comparison to poly(vinylidene fluoride-co-hexafluoropropene) (PVDF) as reference material, which is established as coating material of DES in clinical applications. Our results show that adhesion could be improved for HUVEC on PDC, PPDO and PEI compared to PVDF, whereas almost no SMC attached to the scaffolds indicating a cell-specific response of HUVEC towards the different fibrous structures. Proliferation and apoptosis results revealed that PPDO and PEI have no significant negative influence on vitality and cell cycle behaviour compared to PVDF. Hence, they represent promising candidates for temporary blood vessel support that induce HUVEC attachment and prevent SMC proliferation.

Enhanced Cellular Function of Human Vascular Endothelial Cell on Poly (ε-caprolactone)/Gelatin Coaxial-Electrospun Scaffold

An extracellular matrix-like poly (ε-caprolactone) (PCL)/gelatin core-shell nanofibrous scaffold with high hydrophilicity was developed by coaxial-elecrospinning. However, the behavior of vascular endothelial cells (ECs) on the modified scaffold remains limited. In this study, human umbilical vein ECs (HUVECs) were seeded onto PCL scaffolds with or without gelatin. Morphological changes of HUVECs were observed under confocal laser scanning microscopy (LSCM). HUVECs’ adhesion, proliferation and apoptosis were detected by MTT assay and flow cytometry (FCM). Our results showed that HUVECs on PCL/gelatin scaffolds with identical polygonal and cobblestone-like characteristics reached confluence after 7 days. Modification of PCL nanofibers significantly promoted the attachment of HUVECs onto scaffolds within 1 hour. Compared to pristine PCL, a two-fold increase in proliferation of HUVECs was also observed after 7 days, whereas the apoptosis of HUVECs was obviously reduced by 40% on the modified scaffolds. In summary, these results indicated modified PCL/gelatin scaffold developed by coaxial-elecrospinning can increase the adhesion, proliferation, and suppress apoptosis of HUVECs, suggesting it has a great potential and promising vascular graft in tissue engineering applications.

Enhanced cellular responses of vascular endothelial cells on poly-γ-glutamic acid/PU composite film treated with microwave-induced plasma at atmospheric pressure

Poly-γ-glutamic acid (γ-PGA), which is produced by microbial fermentation, is a biodegradable, hydrophilic and non-toxic biomaterial. γ-PGA has many carboxyl groups that makes it a polyanionic biopolymer with swelling ability, biocompatibility and anticoagulant activity. On the other hand, few studies have examined the effect of γ-PGA on the cellular activity of human umbilical vein endothelial cells (HUVECs). The present study evaluated the effects of γ-PGA and fabricated γ-PGA/PU composite films on the attachment and proliferation of HUVECs after treatment with microwave plasma at atmospheric pressure. The results confirmed that γ-PGA is capable of increasing the proliferation and differentiation of HUVECs to form capillary tubes with enhanced alignment and organization. Moreover, the microwave plasma modified γ-PGA/PU composite film was more hydrophilic and the surface roughness was enhanced. In addition, the attachment and proliferation of the HUVECs were increased by the plasma treatment. These results suggest that γ-PGA and surface modified γ-PGA/PU composite can be applied as bioactive and biocompatible materials in vascular tissue engineering but further testing of γ-PGA and γ-PGA/PU composite films will be needed to confirm the potential effects for use in vascular applications. Open image in new window

Cellular responses of vascular endothelial cells on surface modified polyurethane films grafted electrospun PLGA fiber with microwave-induced plasma at atmospheric pressure

To improve cell–material interaction, the surface modification of materials in tissue engineering is carried out by change in chemical structure or by ion-beam irradiation and plasma treatment. The modifications of biological surface, such as coating or peptide grafting, may also enhance endothelialization of biomaterial surfaces. In this study, the surface of polyurethane (PU) grafted with electrospun poly ( d, l-lactic-co-glycolic acid) (PLGA) fibers (PU/PLGA) was modified with microwave-induced argon plasma and the PU/PLGA films with plasma modified surface were examined to investigate their effects on the attachment and growth of human umbilical vein endothelial cells (HUVECs) for applications in vascular tissue engineering. In comparison with the control, PU film with microwave plasma modified surface was more hydrophilic and the roughness of the surface was enhanced. Furthermore, the plasma treatment on PU/PLGA has significantly increased the attachment of HUVECs and proliferation of the cells was slightly enhanced as well. These results suggested that the surface of PU/PLGA films modified with plasma treatment can be effectively used as a biocompatible material for vascular tissue engineering application, although more in-depth studies are needed.

The effects of co-culture with fibroblasts and angiogenic growth factors on microvascular maturation and multi-cellular lumen formation in HUVEC-oriented polymer fibre constructs

In the present study, polymer monofilaments were embedded in fibrin seeded with human umbilical vein endothelial cells (HUVEC) to guide HUVEC attachment and migration in order to form oriented vessel-like structures between adjacent monofilaments. Histology and fluorescent fibrin experiments confirmed that microvessel-like structures, which were developing between polymer monofilaments embedded in fibrin, contained a lumen. The effect of human fibroblasts and growth factors (VEGF and bFGF) over the microvessel formation process was tested. The effects of VEGF and bFGF were dose-dependent. The effect of VEGF was optimum at the lower concentration tested (2 ng/mL), while that of bFGF was optimum at the higher tested concentration (20 ng/mL). Furthermore, the use of fibroblasts significantly improved the maturation of the microvessels compared to control and to samples cultured with VEGF and bFGF.

Biological relevance of ion energy in performance of human endothelial cells on ion‐implanted flexible polyurethane surfaces

To improve the biocompatibility of polyurethane (PUR), we modified the surface by irradiation with different ions (Carbon; C, Oxygen; O, Nitrogen; N, or Argon; Ar) at 0.3-50 keV energy and doses of 1,00E+13 - 1,00E+15 ions/cm(2). The effects of ion implantation using different ion energies and densities were observed on adhesion, proliferation, and viability of human umbilical vein endothelial cells (HUVECs). The long-term in vitro stability of ion-implanted PUR was also investigated. Ion irradiation moderately affected the surface roughness (R(a)), but strongly enhanced the work of adhesion (W(a)). Cell adhesion was markedly improved on O-, N-, and Ar-, but not on C-implanted PUR surfaces. Medium ion energies and lower ion doses produced the best HUVEC attachment and proliferation, indicating the importance of choosing the proper range of energy applied during ion irradiation. In addition, apoptosis rates were significantly reduced when compared with unmodified PUR (uPUR). N implantation significantly protected the surface, although C implantation led to stronger surface erosions than on uPUR. In total, ion implantation on flexible PUR surfaces strongly improved the material surface characteristics and biocompatibility. Electron beam ion implantation within an appropriate energy window is thus a key to improving flexible PUR surfaces for clinical use to support endothelial cell performance. Thus, it can contribute to designing small-diameter grafts, which are in great demand, towards vascular tissue engineering applications.

Co-assembling peptides as defined matrices for endothelial cells

Self-assembling peptides and peptide derivatives bearing cell-binding ligands are increasingly being investigated as defined cell culture matrices and as scaffolds for regenerative medicine. In order to systematically refine such scaffolds to elicit specific desired cell behaviors, ligand display should ideally be achieved without inadvertently altering other physicochemical properties such as viscoelasticity. Moreover, for in vivo applications, self-assembled biomaterials must exhibit low immunogenicity. In the present study, multi-peptide co-assembling hydrogels based on the β-sheet fibrillizing peptide Q11 (QQKFQFQFEQQ) were designed such that they presented RGDS or IKVAV ligands on their fibril surfaces. In co-assemblies of the ligand-bearing peptides with Q11, ligand incorporation levels capable of influencing the attachment, spreading, morphology, and growth of human umbilical vein endothelial cells (HUVECs) did not significantly alter the materials' fibrillization, β-turn secondary structure, or stiffness. RGDS-Q11 specifically increased HUVEC attachment, spreading, and growth when co-assembled into Q11 gels, whereas IKVAV-Q11 exerted a more subtle influence on attachment and morphology. Additionally, Q11 and RGDS-Q11 were minimally immunogenic in mice, making Q11-based biomaterials attractive candidates for further investigation as defined, modular extracellular matrices for applications in vitro and in vivo.

N-Myc Downstream-Regulated Gene 2 Induces Angiogenesis

Angiogenesis is the process of generating new capillary blood vessels from pre-existing vessels and a fundamental process in the normal development, reproduction, and wound healing. Since neovascularization in physiological processes is tightly regulated by a balance of angiogenic and anti-angiogenic factors, disrupted balance effect plays a leading role in the progress of diseases such as tumor growth, rheumatoid arthritis, and various blood vesselrelated pathology. Tumor growth relies on angiogenesis to receive an adequate supply of nutrients and oxygen. In addition, the newly formed blood vessels provide a way for tumor cells to enter the circulation and to metastasize to distant organs. Therefore, treatment of cancer with antiangiogenic agents will control cancer cell growth and metastasis. Among a variety of angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have a prominent activity in tumor metastasis and mortality. Specially, VEGF expression is induced in various cancers by several stimuli and its transcriptional activation is mediated by hypoxia-inducible factor-1 (HIF1). Since angiogenesis requires a multi-step mechanism, the process is mediated by a wide range of angiogenic regulators. In an effort to search for endogenous angiogenic factors, we found that N-myc downstream-regulated gene 2 (NDRG2) is involved in angiogenesis. Human NDRG2 belongs to a family of NDRG. The human NDRG family includes four identified members, NDRG1, NDRG2, NDRG3, and NDRG4, which are highly homologous except for in the Cand N-terminal regions. NDRG2 initially identified as a candidate tumor suppressor gene since its expression is reduced in several cancer cells. It has also been suggested that NDRG2 may have roles in cell differentiation, Alzheimer’s disease, and neurite overgrowth. However, NDRG2 activity related to angiogenesis was not yet reported. Since endothelial cell migration through extracellular matrix is one of essential steps for neovascularization, we tested the effect of conditioned medium derived from NDRG2-transfected cells (NDRG2-CM) on endothelial cell migration. To establish cell line expressing NDRG2, human embryonic kidney (HEK) 293 cells were transfected with NDRG2 expression plasmid and transfected cells stably expressing NDRG2 were selected as described in experimental section. After expression of NDRG2 was confirmed by Western blotting analysis, conditioned medium (CM) from the selected cells was obtained. In chemotaxis chamber, human umbilical vein endothelial cells (HUVECs) were treated with CM from control transfected cells and stably transfected cells for 2 h and HUVECs that had been migrated through membrane pores were counted under microscope. NDRG2-CM showed significantly induced migratory effect, compared to control-CM (Fig. 1A and B). Therefore, these data indicate that NDRG2-CM has the biological activity that can induce endothelial cell migration. We next examined the ability of NDRG2-CM to promote the mesh-like structures of HUVECs on matrigel. Matrigel is generally used for studying HUVEC attachment, migration and differentiation. The extent of mesh-like structure was more enhanced in HUVECs influenced in NDRG2-CM than that of control CM (Fig. 2A and B). This result indicates that NDRG2-CM has the ability of inducing tube formation of endothelial cells. We then measured the protein level of VEGF in NDRG2-

Assessment of fibronectin conformation adsorbed to polytetrafluoroethylene surfaces from serum protein mixtures and correlation to support of cell attachment in culture

Surfaces of polytetrafluoroethylene (PTFE) were exposed to buffered aqueous solutions containing radio-labeled human fibronectin ([125I]Fn), Fn/bovine serum albumin (BSA) binary mixtures of various ratios or whole human plasma dilutions for 1 h. Total adsorbed Fn and albumin adsorption following rinsing was quantified on this surface. 125I-labeled monoclonal antibodies against either the tenth type-III Fn repeat unit (containing the cell-binding RGDS integrin recognition motif) or the Fn amino-terminal domain were used to probe the accessibility of each of these respective Fn regions post-adsorption. Human umbilical vein endothelial cells (HUVECs) were cultured on PTFE surfaces pre-exposed to each of these protein adsorption conditions and compared to identical conditions on tissue culture polystyrene (TCPS). Fn adsorption to PTFE is dependent upon the concentration of albumin co-adsorbing from solution: albumin out-competes Fn for PTFE surface sites even at non-physiological Fn/HSA ratios 10–100-fold biased in Fn. Antibodies against Fn do not readily recognize Fn adsorbed on PTFE as the HSA co-adsorption concentration in either binary mixtures or in plasma increases, indicating albumin masking of adsorbed Fn. At Fn/HSA ratios rich in Fn (1 : 1, 1 :100), albumin co-adsorption actually improves anti-Fn antibody recognition of adsorbed Fn. HUVEC attachment efficiency to PTFE after protein adsorption correlates with amounts of Fn adsorbed and levels of anti-Fn antibody recognition of Fn on PTFE, linking cell attachment to integrin recognition of both adsorbed Fn density and Fn adsorbed conformation on PTFE surfaces.

Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan-collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require precoating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

Effects of radiofrequency glow discharge and oligopeptides on the attachment of human endothelial cells to polyurethane.

Activation of the surface of small diameter polyurethane vascular grafts using radiofrequency glow discharge (RFGD) and covalent linkage of cell attachment oligopeptides may improve graft patency. The effects of RFGD treatment were investigated on a polyurethane-polydimethylsiloxane (PU-PDMS) copolymer (Cardiothane-51; Kontron Cardiovascular, Inc., Everett, MA) membrane fabricated using the spray, phase-inversion technique. RFGD using H2O vapor was used to functionalize the membrane surface, reacted with 1',1 carbonyldiimidazole and covalently bound with oligopeptides (RGDS, RGES). Membranes not subjected to RFGD, either unmodified or with adsorbed fibronectin (Fn), vitro-nectin (Vn), RGDS, RGDV, and RGES, were used as controls. RFGD treated membrane surfaces were evaluated using electron spectroscopy for chemical analysis, which demonstrated a qualitative increase in nitrogen and silicon compared to unmodified PU-PDMS. Indirect confirmation of surface hydroxylation was provided by metallization with palladium and nickel, demonstrating uniform metallization of RFGD treated PU-PDMS surfaces. Human umbilical vein endothelial cells (HUVEC) were seeded at a density of 10(4) cells/cm2 and cell attachment assessed at 3 hr, 24 hr, and 7 days. Untreated PU-PDMS and membrane with adsorbed oligopeptides demonstrated poor HUVEC attachment at all intervals. Adsorbed Fn and Vn had significantly better early cell attachment and growth (p < 0.01). RFGD improved initial attachment and growth over non-RFGD treated controls (p < 0.01) for RGDS bound membranes, which performed as well as Fn controls (N.S.). PU-PDMS membranes can be modified using RFGD to covalently link oligopeptides. RFGD treatment alone, or with covalent linkage of cell attachment oligopeptides, improves HUVEC attachment and growth in a static environment.

Characterization of Endothelial Cell Differential Attachment to Fibrin and Fibrinogen and Its Inhibition by Arg-Gly-Asp-Containing Peptides

We investigated the adhesion of human umbilical vein endothelial cells (HUVECs) to fibrin(ogen) molecule of varying structure for identifying sites that mediate cell attachment. Fibrin was prepared either with ancrod which liberates only FPA from fibrinogen, or with thrombin, which liberates both FPA and FPB. Both fibrin preparations equally supported HUVEC attachment. GRGDS, RGD-containing peptides of snake venoms, and monoclonal antibodies against alpha v beta 3 (23C6 and 7E3) inhibited the attachment of HUVECs to fibrin by 65-75%. In contrast, the attachment of HUVECs to fibrinogen was less effective and was almost completely inhibited by both RGD-containing peptides and by antibodies against integrin alpha v beta 3 (85-95% inhibition). The C-terminal dodecapeptide of fibrinogen gamma chain (residues 400-411) inhibited minimally the attachment of HUVECs to fibrin. Additionally, the binding of RGD-containing snake venom peptides to HUVECs was both RGD- and divalent-cation-dependent. The IC50S for inhibition of HUVEC attachment to fibrin were 0.09 microM (rhodostomin), 1.54 microM (trigramin) and 1.64 microM (halysin). These results indicate that fibrin mediated support of cell attachment is independent of the cleavage of FPB from fibrinogen. HUVEC attachment to fibrinogen was almost completely inhibited by RGD-containing peptides and by antibodies against alpha v beta 3. In contrast, the attachment to fibrin was partially resistant to RGD-containing peptides and to the monoclonal antibodies against integrin alpha v beta 3. However, alpha v beta 3 is the major receptor mediating HUVEC attachment to fibrin.

Reactivity of Synthetic Peptide Analogs of Adhesive Proteins in Regard to the Interaction of Human Endothelial Cells With Extracellular Matrix

Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion sites” of fibronectin, vitronectin, von Willebrand factor, and α-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the γ-chain of human fibrinogen sequence 400-411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys-Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.

Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix

Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion site” of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400–411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys- Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His- His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.