Endothelial Extracellular Matrices Research Articles

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices' defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices' defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

Human Platelet-Rich Plasma- and Extracellular Matrix-Derived Peptides Promote Impaired Cutaneous Wound Healing In Vivo

Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds. Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment. Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.

IMPACTS OF LOW DENSITY LIPOPROTEIN ON ADHESIVE MECHANICAL PROPERTIES OF ENDOTHELIAL CELLS AND COLLAGEN IV

Cell adhesion refers to the adhesion between cells and cells, cells and extracellular matrices (ECMs). Adhesion is a main communication pathway in cell–cell and cell–ECM interactions. Higher concentrations of low density lipoprotein (LDL) may decrease the adhesive forces between endothelial cells and ECMs in blood vessels. This study aimed to investigate the influence of LDL on the adhesive mechanical properties between endothelial cells (ECs) and ECMs by using the micropipette aspiration technique and flow chambers. Human umbilical vein endothelial cells (HUVECs) were used in this study and the LDL was obtained from fresh human plasma. For micropipette aspiration experiments, ECs were digested into single-cell suspensions and were inoculated in the flow chamber, which was coated with 2 μg/ml of Collagen IV (Col IV) mixed with LDL at four concentrations of 0, 50, 100 and 150 μg/ml, respectively. The micropipette aspiration experiment started 30 min after incubation. For flow chamber experiments, the same mixture was applied on the coverslips. ECs were perfused by three different kinds of shear stress in the flow chamber of 12.0, 24.0, 36.0 dynes/cm2, respectively. The results indicated that the adhesive force of ECs on Col IV-coated chamber decreased as LDL concentration increased (P < 0.05). Furthermore, in the flow conditions, the adhesive-retention ratio of cells was significantly decreased as shear stress and LDL concentration increased (P < 0.05). The results indicated that the adhesive force between ECs and ECMs was dependent on LDL concentration. Higher LDL concentration results in lower adhesive force. The adhesive-retention ratio of cells was relevant to the effect of shear stress and the alteration of shear stress that could influence the adhesion of cells. Similar study may potentially aid to understand the results of future pharmacological studies in atherosclerosis.

Author Response: Effects of Fibroblastic and Endothelial Extracellular Matrices on Corneal Endothelial Cells

Author Response: Effects of Fibroblastic and Endothelial Extracellular Matrices on Corneal Endothelial Cells

Effects of Fibroblastic and Endothelial Extracellular Matrices on Corneal Endothelial Cells

Extracellular matrices (ECM) isolated from chick embryo fibroblast and human and rabbit corneal stromal cells induce polarization and elongation of corneal endothelial cells in culture. ECM isolated from rabbit corneal or bovine aortic endothelial cells neither polarize nor elongate corneal endothelial cells in culture. By indirect immunofluorescence, fibronectin is seen as arrays of long fibers in fibroblastic ECM, whereas in endothelial ECM, fibronectin is found in discreet foci as short fibers. The morphology corneal endothelial cells in culture is associated with the structure of the ECM laid down; short fibers in clusters associated with a typical polygonal shape, long polarized fibers inducing a fibroblastic-like appearance. ECM isolated from both fibroblastic or endothelial sources promote cell growth. Since coating the plastic with plasma fibronectin or denatured collagen does not promote endothelial cell growth, the authors assume the polymerized fibronectin network or a component bound to this network enhances the growth of corneal endothelial cells cultured in the presence of serum.

Phenotypic expression of marrow cells when grown on various substrata

Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen I. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function.

Platelet and Shear Rate Promote Tumor Cell Adhesion to Human Endothelial Extracellular Matrix - Absence of a Role for Platelet Cyclooxygenase

Blood platelets are thought to be involved in certain aspects of malignant dissemination. To study the role of platelets in tumor cell adherence to vascular endothelium we performed studies under static and flow conditions, measuring tumor cell adhesion in the absence or presence of platelets. We used highly metastatic human adenocarcinoma cells of the lung, cultured human umbilical vein endothelial cells (ECs) and extracellular matrices (ECM) prepared from confluent EC monolayers. Our results indicated that under static conditions platelets do not significantly increase tumor cell adhesion to either intact ECs or to exposed ECM. Conversely, the studies performed under flow conditions using the flat chamber perfusion system indicated that the presence of 2 x 10(5) pl/microliters in the perfusate significantly increased the number of tumor cells adhered to ECM, and that this effect was shear rate dependent. The maximal values of tumor cell adhesion were obtained, in presence of platelets, at a shear rate of 1,300 sec-1. Furthermore, our results with ASA-treated platelets suggest that the role of platelets in enhancing tumor cell adhesion to ECM is independent of the activation of the platelet cyclooxygenase pathway.

Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment.

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.

The interaction of melanoma cells with fibroblasts and endothelial cells in three-dimensional macromolecular matrices: a model for tumour cell invasion.

Comparative quantitative data are presented concerning the adhesion, proliferation and invasive behaviour of RPMI-3460 melanoma cells on (1) plain collagen gels, (2) monolayer cultures of fibroblasts and endothelial cells growing on the gel surface, and (3) the exposed endothelial and fibroblast extracellular matrices (ECMs). Both types of ECMs enhanced melanoma cell adhesion and proliferation (compared with plain gels) and had marked, but distinctive, effects on melanoma morphology. The thickness and composition of the ECMs was altered by treatment of the matrices with enzymes (trypsin, elastase and chondroitinase ABC) or by using ECMs produced by endothelial cells at various times after confluence. Variations in the thickness and composition of the ECMs had no effect on the behaviour of melanoma cells growing on these matrices; our results suggest that the glycoproteins and glycosaminoglycan ECM constituents removed by digestion with the enzymes do not play an important role in melanoma cell attachment, proliferation and migration. Melanoma cells plated on the surface of a plain collagen gel rapidly migrated down into the collagen matrix, with approximately 30% of the cells found within the gel after 6 days of incubation. Fibroblast and endothelial ECMs significantly and distinctively inhibited melanoma invasion into the underlying collagen gel. The extensive invasion of melanoma cells into the gel was not accompanied by hydrolysis of the collagen fibres. Conversely, fibroblast and endothelial ECMs, which acted as effective barriers, were extensively hydrolysed by the melanoma cells. The possible use of ECMs deposited on collagen in the study of melanoma local invasion (on fibroblast ECMs) and extravasation (on endothelial ECMs) is discussed.

Cell interaction with the extracellular matrices produced by endothelial cells and fibroblasts

The extracellular matrices (ECM) produced by cultured bovine corneal endothelial cells and chick embryo fibroblasts were compared for their induction of cell attachment, proliferation and differentiation. The corneal endothelial ECM (cECM) induced a comparable and rapid attachment and flattening of both human Ewing's sarcoma and colon carcinoma cells which utilize fibronectin and laminin as adhesive glycoproteins, respectively. In contrast, the ECM produced by fibroblasts (fECM) readily supported the attachment and flattening of Ewing's sarcoma cells but had only a small effect on the carcinoma cells. Vascular endothelial cells were stimulated to proliferate by both types of matrices, but to a lesser extent by the fECM. In contrast, the formation of a closely apposed, non-overlapping and contact-inhibited endothelial cell monolayer was only dictated by the cECM. Vascular endothelial cells cultured on fECM grew on top of each other and incorporated [ 3H]thymidine even late at confluency. Neunte outgrowth (ciliary ganglion cells) and network formation (adult rat oligodendrocytes) were promoted by both types of matrices but in a more consistent manner with the cECM. It is likely that the small amounts of laminin deposited by chick embryo fibroblasts into their ECM are responsible for its efficient induction of neunte outgrowth and for the limited degree of carcinoma cell attachment and flattening. It is thus demonstrated that differences in chemical composition and supramolecular arrangement between cECM and fECM result not only in differences in the attachment, spreading and proliferative responses of cells but also in the expression of their characteristic morphological appearance and differentiated functions.

Altered adhesiveness of tumor cell surface variants with reduced metastasizing capacity--reduced adhesiveness to vascular wall components in culture.

Heterotypic adhesiveness of surface variant clones of B-16 melanoma cells exhibiting different metastasizing capacity was studied with respect to components of the vascular wall in culture including endothelial (E) and smooth muscle (SM) cells from adult bovine aorta, and the extracellular matrices laid down by them. The ricin-resistant cells (ricinR) and the wheat-germ agglutinin-resistant (WGAR) cells, both of which showed reduced hematogenous metastases in vivo, showed reduced adhesiveness specifically to endothelial cells and extracellular matrices. This reduced heterotypic adhesiveness is in contrast to the homotypic adhesive properties in which respect the ricinR cells had similar values to the parental cells, while the WGAR cells had much higher values than the latter. There appeared to be a positive correlation between metastasizing capacity and specific adhesiveness to E cells and the extracellular matrix surfaces.