Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

Differential mitogenic responses of human macrovascular and microvascular endothelial cells to cytokines underline their phenotypic heterogeneity

A variety of growth factors promote the complex multistep process of angiogenesis. The mitogenic activity of vascular endothelial growth factors (VEGFs) and placental growth factors (PlGFs), known as cytokines acting predominantly on endothelial cells, was tested on human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (MIEC) and compared with the potency of the universally acting basic fibroblast growth factor (FGF-2). The cells were seeded at different cell numbers and incubated with various doses of growth factors for a period of 24-72 h in culture medium +/- serum. Proliferation was determined by measuring the optical density after staining the cells with the tetrazolium salt WST-1. VEGF121 and VEGF165 increased the number of HUVEC and MIEC at low and high seeding densities various doses and incubation times. The efficiency of FGF-2 was less pronounced at high seeding densities of the cells under serum-free conditions. PlGF-1 and PlGF-2 stimulated mitogenesis on HUVEC only at low cell numbers and after a short incubation time by 125 +/- 3% and 102 +/- 5% (P < 0.001), respectively. Longer incubation times with the lower seeding density in the absence of FCS did not induce a significant stimulatory effect of the PlGFs. MIEC responded stronger to all growth factors. In particular under serum free conditions, PlGF-1 and PlGF-2 effectively stimulated cell proliferation by 247 +/- 54% (P < 0.01) and 288 +/- 40% (P < 0.05) at low cell numbers, and by 81 +/- 13% (P < 0.05) and 49 +/- 13% (P < 0.01), respectively, at high cell numbers. The addition of fetal calf serum caused a reduced proliferative response of all growth factors on both cell types related to the controls. In conclusion, MIEC and HUVEC differ in their proliferative response to VEGFs, PlGFs and FGF-2.     

Bacterial adherence to human endothelial cells

The adult respiratory distress syndrome (ARDS) is frequently caused by exposure of the lung endothelium to circulating endotoxin (lipopolysaccharide, LPS) and pulmonary infections frequently develop during the course of ARDS. The present studies demonstrate that LPS and interleukin 1 (IL-1, a mediator released by endothelial cells after exposure to LPS) enhance the adherence of Staphylococcus aureus to human umbilical vein endothelial cells. gamma-Interferon, another mediator that induces expression of some cell surface antigens on endothelial cells, had no effect on bacterial adherence. The adherence of bacteria to endothelium was increased by prior opsonization of the bacteria with fresh human serum and was reduced by prior absorption of the serum with bacteria before the use of the serum for opsonization. The capacity of LPS to increase bacterial adherence was time dependent and was maximally expressed after 6 h of exposure; it was blocked by exposure of endothelial cells to LPS in the presence of reduced temperature or dactinomycin (Actinomycin D). These observations suggest that circulating LPS not only can trigger the development of ARDS but also may predispose the lung to the development of pulmonary infections by increasing adherence of bacteria to endothelium.     

Contrasting effects of inflammatory stimuli on neutrophil and monocyte adherence to endothelial cells

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydraulic conductance of pulmonary microvascular and macrovascular endothelial cell monolayers

Endothelial cells isolated from pulmonary arteries (RPAEC) and microcirculation (RPMVEC) of rat lungs were grown to confluence on porous filters and mounted on an Ussing-type chamber. Transmembrane pressure (deltaP) was controlled by the reservoir height, and the filtration rate corrected for surface area (J(v)/A) was measured by timing fluid movement in a calibrated micropipette. These parameters were used to calculate hydraulic conductance (Lp) by using linear regression of J(v)/A on deltaP. Mean Lp values for newly confluent RPAEC monolayers were 22 times higher than those for RPMVEC monolayers (28.6 +/- 5.6 vs. 1.30 +/- 0.50 x 10(-7) cm x s(-1) x cmH2O(-1); P < or = 0.01). After confluence was reached, electrical resistance and Lp remained stable in RPAEC but continued to change in RPMVEC with days in culture. Both phenotypes exhibited an initial time-dependent sealing response, but Lp also had an inverse relationship to deltaP in RPMVEC monolayers > or = 4 days postconfluence that was attributed to cell overgrowth rather than junctional length. In a comparison of the cadherin contents, E-cadherin was predominant in RPMVEC, but VE-cadherin was predominant in RPAEC. At a constant deltaP of 40-45 cmH2O for 2 h, J(v)/A increased 225% in RPAEC monolayers but did not change significantly in RPMVEC monolayers. Significant decreases in Lp were obtained after treatment with 5% albumin, GdCl3, or isoproterenol plus rolipram in both phenotypes. Thus lung microvascular endothelial cells exhibited a significantly lower Lp than conduit vessel endothelium, which would limit alveolar flooding relative to perivascular edema cuff formation during increased pulmonary vascular pressures.     

Group B streptococcal pilus proteins contribute to adherence to and invasion of brain microvascular endothelial cells

Surface filamentous structures known as pili have been discovered recently in the gram-positive streptococcal pathogens that cause invasive disease in humans, including group B Streptococcus (GBS). We show that two GBS proteins involved in pilus formation, encoded by pilA and pilB, also facilitate the interaction of this important agent of central nervous system infection with endothelial cells of the human blood-brain barrier.     

Lysoplasmenylcholine increases neutrophil adherence to human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in release of choline lysophospholipids [lysophosphatidylcholine (lysoPtdCho) and lysoplasmenylcholine (lysoPlsCho)]. These amphiphilic metabolites have been implicated in arrhythmogenesis following the onset of myocardial ischemia, but studies examining their direct effects on the vasculature remain limited. We and others have shown that thrombin and lysoPtdCho can increase cell surface adhesion molecules and adherence of circulating inflammatory cells to the endothelium. This study supports our hypothesis that these changes may be mediated, at least in part, by lysoPlsCho, thus implicating this metabolite as an inflammatory mediator in the coronary vasculature and a modulator of the progression of atherosclerosis. Apical stimulation of HCAEC with thrombin resulted in the production and release of choline lysophospholipids from the apical surface of the HCAEC monolayer. Basolateral stimulation had no effect on choline lysophospholipid production or release from either the apical or basolateral surface of the HCAEC monolayer. Incubation of HCAEC with lysoPlsCho or lysoPtdCho resulted in similar increases in HCAEC surface expression of P-selectin and E-selectin. Furthermore, lysoPlsCho increased cell surface expression of P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 with a time course similar to that of thrombin stimulation. Increased presence of cell surface adhesion molecules may contribute to the significant increase in adherence of neutrophils to either thrombin- or lysoPlsCho-stimulated HCAEC. These results demonstrate that the presence of thrombin at sites of vascular injury in the coronary circulation, resulting in increased choline lysophospholipid release from the HCAEC apical surface, has the potential to propagate vascular inflammation by upregulation of adhesion molecules and recruitment of circulating inflammatory cells to the endothelium. endothelium; arrhythmogenesis; inflammation; lysophospholipids     

Spontaneous immortalization of human dermal microvascular endothelial cells

AIM: To establish and characterize a spontaneously immortalized human dermal microvascular endothelial cell line, iHDME1.METHODS: We developed a spontaneous immortalization method. This approach is based on the application of optimized culture media and culture conditions without addition of any exogenous oncogenes or carcinogens. Using this approach, we have successfully established a microvascular endothelial cell line, iHDME1, from primary human dermal microvascular endothelial cells. iHDME1 cells have been maintained in culture dishes for more than 50 passages over a period of 6 mo. Using a GFP expressing retrovirus, we generated a GFP-stable cell line (iHDME1-GFP).RESULTS: iHDME1 retain endothelial morphology and uniformly express endothelial markers such as VEGF receptor 2 and VE-cadherin but not α-smooth muscle actin (α-SM-actin) and cytokeratin 18, markers for smooth muscle cells and epithelial cells respectively. These cells retain endothelial properties, migrate in response to VEGF stimulation and form 3-D vascular structures in Matrigel, similar to the parental cells. There is no significant difference in cell cycle profile between the parental cells and iHDME1 cells. Further analysis indicates enhanced stemness in iHDME1 cells compared to parental cells. iHDME1 cells display elevated expression of CD133 and hTERT.CONCLUSION: iHDME1 cells will be a valuable source for studying angiogenesis.     

Increased adherence of oxidant-treated human and bovine erythrocytes to cultured endothelial cells

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1-3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30-33%) in the external leaflet of the bilayer and a concomitant increased (four- to five-fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross-linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15-20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant-induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant-treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC-540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)-treated erythrocytes compared to 30% of untreated RBCs exhibited MC-540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant-treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross-linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte-endothelial cell interaction.     

Cancer cells regulate biomechanical properties of human microvascular endothelial cells

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1(high) cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs.     

Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells

Angiogenesis is one of the most recent physiological functions attributed to products of cytochrome P-450 (CYP450) enymes. To test this at a molecular level in human cells, we used a cloned cDNA for the human endothelial enzyme CYP450 2C9 (CYP2C9) to study growth as well as differentiation of human microvascular endothelial cells from the lung (HMVEC-L). Using adenoviral vectors overexpressing mRNA for CYP2C9, we show that the presence of CYP2C9 doubles thymidine incorporation and stimulates proliferation of primary cultures of endothelial cells compared with Ad5-GFP (control) in 24 h. In addition, there is a significant increase of tube formation in Matrigel after infection of HMVEC-L with Ad5-2C9 than with Ad5-GFP. More interestingly, Ad5-2C9 expressing the antisense product of CYP2C9 (2C9AS) inhibited tube formation compared with both Ad5-GFP as well as the Ad5-2C9 constructs. Finally, we tested the most abundant arachidonic acid metabolite of CYP2C9, 14,15-epoxyeicosatrienoic acid, which induced angiogenesis in vivo when embedded in Matrigel plugs and implanted in adult rats. These data support an important role for CYP2C9 in promoting angiogenesis.     

Human monocyte adherence: a primary effect of chemotactic factors on the monocyte to stimulate adherence to human endothelium

Monocyte emigration into areas of inflammation is initiated by monocyte adherence to the microvascular endothelium which may be induced by the local production of chemotactic factors at the inflammatory site. However, it is not clear whether such stimuli act on the monocyte and/or the endothelial cell to promote this effect. Accordingly, the effect of the chemotactic peptides C5a des arg and formyl-methionyl-leucyl-phenylalanine (FMLP) on human monocyte adherence to human microvascular endothelial cell monolayers was investigated in vitro. Monocytes (92 to 98% pure) were isolated by discontinuous plasma-Percoll density gradients and cell elutriation, methods designed to minimize monocyte exposure to endotoxin. Mean spontaneous (unstimulated) adherence of 111Indium-tropolonate-radiolabeled monocytes to microvascular endothelial cell monolayers was 19.7% +/- 1.3. Monocyte adherence to microvascular endothelial cell monolayers was stimulated in a dose-response fashion in the presence of C5a des arg or FMLP to a maximum mean adherence of 47.2% +/- 2.9 or 43.8% +/- 2.2, respectively. C5a des arg or FMLP stimulated monocytes to adhere to monolayers of human vascular smooth muscle cells, human dermal fibroblasts, or serum-coated plastic wells in a comparable fashion as to endothelial cells. The simultaneous presence of both chemotactic peptides C5a des arg and FMLP in the assay system stimulated monocyte adherence to the same degree as either stimulus alone. This finding suggested that those monocytes stimulated to adhere by C5a des arg were the same subpopulation responding to FMLP. Spontaneous monocyte adherence (in the absence of chemotactic peptides) to both endothelial cell monolayers and serum-coated plastic wells was reduced in the presence of plasma, but chemotactic peptides induced a significant, albeit reduced, adhesion of monocytes in this circumstance. The pretreatment of monocytes with either C5a des arg or FMLP prior to the adherence assay induced stimulus-specific desensitization of monocyte adherence. Neither a desensitization nor stimulated monocyte adherence occurred when endothelial cell monolayers or serum-coated plastic wells were pretreated with either of the chemotactic peptides. The fixation of endothelial cell monolayers prior to the adherence assay did not alter the degree of spontaneous, C5a des arg-stimulated, or FMLP-stimulated monocyte adherence. These data suggest that the stimulated adhesion of monocytes to endothelial cells by C5a des arg or FMLP represents primarily an effect of these chemotactic peptides on the monocyte.     

Chlamydia pneumoniae-infected monocytes exhibit increased adherence to human aortic endothelial cells

Interactions between monocytes and endothelial cells play an important role in the pathogenesis of atherosclerosis, and monocyte adhesion to arterial endothelium is one of the earliest events in atherogenesis. Work presented in this study examined human monocyte adherence to primary human aortic endothelial cells following monocyte infection with Chlamydia pneumoniae, an intracellular pathogen associated with atherosclerosis by a variety of sero-epidemiological, pathological and functional studies. Infected monocytes exhibited enhanced adhesion to aortic endothelial cells in a time- and dose-dependent manner. Pre-treatment of C. pneumoniae with heat did not effect the organism's capacity to enhance monocyte adhesion, suggesting that heat-stable chlamydial antigens such as chlamydial lipopolysaccharide (cLPS) mediated monocyte adherence. Indeed, treatment of monocytes with cLPS was sufficient to increase monocyte adherence to endothelial cells, and increased adherence of infected or cLPS-treated monocytes could be inhibited by the LPS antagonist lipid X. Moreover, C. pneumoniae-induced adherence could be inhibited by incubating monocytes with a mAb specific to the human beta 2-integrin chain, suggesting that enhanced adherence resulted from increased expression of these adhesion molecules. These data show that C. pneumoniae can enhance the capacity of monocytes to adhere to primary human aortic endothelial cells. The enhanced adherence exhibited by infected monocytes may increase monocyte residence time in vascular sites with reduced wall shear stress and promote entry of infected cells into lesion-prone locations.     

Lipid peroxidation and growth inhibition of human microvascular endothelial cells

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid-reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n - 3), arachidonic acid (C20:4. n - 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.     

Regulation of sterol transport in human microvascular endothelial cells

In cultured human dermal microvessel endothelial cells, the rate of efflux (about twofold greater than for fibroblasts under equivalent conditions) was coupled to an equivalent high rate of sterol net transport from the cells to the medium. This net transport was linked with esterification via lecithin:cholesterol acyltransferase. Since the use of free sterol by plasma transferase is constant, such increased net transport indicates that endothelial cells are highly efficient, in competition with plasma lipoproteins, in supplying free sterol for esterification. These results indicate the marked ability of endothelial cells to regulate and maintain their sterol balance in the face of high sterol levels to which these cells are uniquely exposed in human plasma.     

Cultivation of microvascular endothelial cells from human preputial skin

Summary A procedure is described for the isolation and cultivation of microvascular endothelium from human skin. Neonatal foreskins are pooled, washed, minced, and dissociated by a mixture of collagenase and dispase. Microvascular endothelium, liberated in the form of intact capillary fragments, is incompletely separated from fibroblasts and epidermal cells by sieving through nylon mesh, followed by velocity sedimentation on 5% bovine serum albumin. The endothelium-enriched fraction has been maintained in primary culture for up to 3 weeks. The resulting epithelioid colonies have been characterized morphologically by both light and transmission electron microscopy and manifest all of the structural features that distinguish other, large-vessel endothelia in culture. In addition, immunohistochemical studies using an indirect fluorescent antibody technique demonstrate that these cells contain the endothelium-specific product, Factor VIII antigen. This work was supported by National Institutes of Health Grants AM18904 and AM20571, the RGK Foundation, the Charlotte and Sidney Lifschultz Foundation, the Juvenile Diabetes Foundation, and the South Carolina Geenral Medical Faculty Research Appropriation.