Human Dermal Microvascular Cells Research Articles

Expression Profile of CD157 Reveals Functional Heterogeneity of Capillaries in Human Dermal Skin

CD157 acts as a receptor, regulating leukocyte trafficking and the binding of extracellular matrix components. However, the expression pattern and the role of CD157 in human blood (BEC) and the lymphatic endothelial cells (LEC) of human dermal microvascular cells (HDMEC), remain elusive. We demonstrated constitutive expression of CD157 on BEC and LEC, in fetal and juvenile/adult skin, in situ, as well as in isolated HDMEC. Interestingly, CD157 epitopes were mostly localized on BEC, co-expressing high levels of CD31 (CD31High), as compared to CD31Low BEC, whereas the podoplanin expression level on LEC did not affect CD157. Cultured HDMEC exhibited significantly higher numbers of CD157-positive LEC, as compared to BEC. Interestingly, separated CD157− and CD157+ HDMEC demonstrated no significant differences in clonal expansion in vitro, but they showed distinct expression levels of cell adhesion molecules, before and after cytokine stimulation in vitro. In particular, we proved the enhanced and specific adherence of CD11b-expressing human blood myeloid cells to CD157+ HDMEC fraction, using an in vitro immune-binding assay. Indeed, CD157 was also involved in chemotaxis and adhesion of CD11b/c monocytes/neutrophils in prevascularized dermo–epidermal skin substitutes (vascDESS) in vivo. Thus, our data attribute specific roles to endothelial CD157, in the regulation of innate immunity during inflammation.

Divergent TCR-Initiated Calcium Signals Govern Recruitment versus Activation of Human Alloreactive Effector Memory T Cells by Endothelial Cells.

Early human allograft rejection can be initiated when circulating human host versus graft Ag-specific CD8 and CD4 effector memory T cells directly recognize MHC class I and II, respectively, expressed on the luminal surface by endothelium lining graft blood vessels. TCR engagement triggers both graft entry (TCR-driven transendothelial migration or TEM) and production of proinflammatory cytokines. Both TCR-driven TEM and cytokine expression are known to depend on T cell enzymes, myosin L chain kinase, and calcineurin, respectively, that are activated by cytoplasmic calcium and calmodulin, but whether the sources of calcium that control these enzymes are the same or different is unknown. Using superantigen or anti-CD3 Ab presented by cultured human dermal microvascular cells to freshly isolated peripheral blood human effector memory T cells under conditions of flow (models of alloantigen recognition in a vascularized graft), we tested the effects of pharmacological inhibitors of TCR-activated calcium signaling pathways on TCR-driven TEM and cytokine expression. We report that extracellular calcium entry via CRAC channels is the dominant contributor to cytokine expression, but paradoxically these same inhibitors potentiate TEM. Instead, calcium entry via TRPV1, L-Type Cav, and pannexin-1/P2X receptors appear to control TCR-driven TEM. These data reveal new therapeutic targets for immunosuppression.

The Activation of Human Dermal Microvascular Cells by Poly(I:C), Lipopolysaccharide, Imiquimod, and ODN2395 Is Mediated by the Fli1/FOXO3A Pathway.

Endothelial cell (EC) dysfunction has been associated with inflammatory and autoimmune diseases; however, the factors contributing to this dysfunction have not been fully explored. Because activation of TLRs has been implicated in autoimmune diseases, the goal of this study was to determine the effects of TLR ligands on EC function. Human dermal microvascular ECs (HDMECs) treated with TLR3 [Poly(I:C)], TLR4 (LPS), and TLR7 (imiquimod) agonists showed decreased proliferation and a reduced total number of branching tubules in three-dimensional human dermal organoid ex vivo culture. In contrast, the TLR9 ligand class C, ODN2395, increased angiogenesis. The antiproliferative effects of TLR3, TLR4, and TLR7 ligands correlated with significant downregulation of a key regulator of vascular homeostasis, Fli1, whereas TLR9 increased Fli1 levels. Furthermore, Poly(I:C) and LPS induced endothelial to mesenchymal transition that was reversed by the pretreatment with TGF-β neutralizing Ab or re-expression of Fli1. We showed that Fli1 was required for the HDMEC proliferation by transcriptionally repressing FOXO3A. In contrast to TLR9, which suppressed activation of the FOXO3A pathway, TLR3, TLR4, and TLR7 ligands activated FOXO3A as indicated by decreased phosphorylation and increased nuclear accumulation. The inverse correlation between Fli1 and FOXO3A was also observed in the vasculature of scleroderma patients. This work revealed opposing effects of TLR9 and TLR3, TLR4, and TLR7 on the key angiogenic pathways, Fli1 and FOXO3A. Our results provide a mechanistic insight into the regulation of angiogenesis by TLRs and confirm a central role of Fli1 in regulating vascular homeostasis.

MiR-184 exhibits angiostatic properties via regulation of Akt and VEGF signaling pathways.

Corneal avascularity is critical for achieving transparency necessary for proper transmission of light to the lens and visual acuity. Although much is known about angiogenesis and angiostasis, the precise regulation of these processes in the cornea is unclear. MicroRNA (miR)-184, the most abundant corneal epithelial miRNA, has been suggested to function in corneal angiostasis by altering VEGF signaling; however, the mechanism(s) underlying this regulation have not been addressed. Using a combination of in vitro and in vivo assays to evaluate angiogenesis, we demonstrated that human limbal epithelial keratinocytes (HLEKs) engineered to overexpress miR-184 secreted lower amounts of angiogenic mitogens. Human dermal microvascular cells exposed to conditioned medium from miR-184-overexpressing HLEKs were less proliferative and failed to seal linear scratch wounds. The in vivo Matrigel plug assay showed that conditioned medium from miR-184-expressing HLEKs elicited a lesser degree of neovascularization compared with controls. We found that miR-184 directly targets and represses the proangiogenic factors, friend of Gata 2 (FOG2), platelet-derived growth factor (PDGF)-β, and phosphatidic acid phosphatase 2b (PPAP2B). FOG2 regulates VEGF expression, whereas PDGF-β and PPAP2B regulate Akt activity. By attenuating both VEGF and Akt signaling, miR-184 acts as a broad-spectrum negative regulator of corneal angiogenesis.-Park, J. K., Peng, H., Yang, W., Katsnelson, J., Volpert, O., Lavker, R. M. miR-184 exhibits angiostatic properties via regulation of Akt and VEGF signaling pathways.

Fli1 deficiency contributes to the suppression of endothelial CXCL5 expression in systemic sclerosis

CXCL5 is a member of CXC chemokines with neutrophilic chemoattractant and pro-angiogenic properties, which has been implicated in the pathological angiogenesis of rheumatoid arthritis and inflammatory bowel diseases. Since aberrant angiogenesis is also involved in the developmental process of systemic sclerosis (SSc), we herein measured serum CXCL5 levels in 63 SSc and 18 healthy subjects and investigated their clinical significance and the mechanism explaining altered expression of CXCL5 in SSc. Serum CXCL5 levels were significantly lower in SSc patients than in healthy subjects. In diffuse cutaneous SSc (dcSSc), serum CXCL5 levels were uniformly decreased in early stage (<1year) and positively correlated with disease duration in patients with disease duration of <6years. In non-early stage dcSSc (≥1year), decreased serum CXCL5 levels were linked to the development of digital ulcers. Consistently, the expression levels of CXCL5 proteins were decreased in dermal blood vessels of early stage dcSSc. Importantly, Fli1 bound to the CXCL5 promoter and its gene silencing significantly suppressed the CXCL5 mRNA expression in human dermal microvascular endothelial cells. Furthermore, endothelial cell-specific Fli1 knockout mice, an animal model of SSc vasculopathy, exhibited decreased CXCL5 expression in dermal blood vessels. Collectively, these results indicate that CXCL5 is a member of angiogenesis-related genes, whose expression is suppressed at least partially due to Fli1 deficiency in SSc endothelial cells. Since Fli1 deficiency is deeply related to aberrant angiogenesis in SSc, it is plausible that serum CXCL5 levels inversely reflect the severity of SSc vasculopathy.

CCL5, CXCL16, and CX3CL1 are associated with Henoch-Schonlein purpura

Chemokines are involved in the pathogenesis of various vascular inflammations. However, information about chemokines in Henoch-Schonlein purpura (HSP) is limited. Herein, we investigated the serum CCL5, CXCL16, and CX3CL1 levels in HSP patients with controls and the ability of sera from HSP patients on chemokine production in human dermal microvascular endothelial cells. Enzyme-linked immunosorbent assay (ELISA) detected serum CCL5, CXCL16, and CX3CL1 levels in patients with HSP. Human dermal microvascular endothelial cell line (HMEC-1) was treated with sera from patients with HSP at different stages, patients with acute spontaneous urticaria, or controls. Serum levels of CCL5, CXCL16, and CX3CL1 were elevated in HSP patients at acute stage, which correlated with the severity of this disease. Sera from patients with active HSP markedly induced CCL5, CXCL16, and CX3CL1 production at both mRNA and protein levels. In addition, patients' sera-stimulated HMEC-1 supernatants enhanced HL-60 or THP-1 cells migration. Furthermore, patients' sera increased the phosphorylation of inhibitor of κB-α (IκBα) and phosphorylation of extracellular signal-regulated kinase (ERK)1/2 protein levels, upregulated the translocation of nuclear factor-κB (NF-κB) p65 to the nucleus. Taken together, we show firstly that CCL5, CXCL16, and CX3CL1 may be involved in the pathogenesis of HSP. Factors present in sera from patients with active HSP may act as an inducer of inflammatory response in HMEC-1 cells and contribute to chemokine production through NF-κB and ERK 1/2 pathways.

Expression and functional significance of adhesion molecules on cultured endothelial cells in response to ionizing radiation.

Upregulation of adhesion molecules on endothelial cells following irradiation has been shown, but the functional significance of this upregulation in various endothelial cell lines is not clear. We have developed an in vitro flow model to study the functional consequences of the radiation-induced upregulation of E-selectin and intercellular adhesion molecule (ICAM-1). Human dermal microvascular endothelial cells (HDMEC), human umbilical vein endothelial cells (HUVEC), or transformed human microvascular endothelial cells (HMEC-1) were grown in 35-mm dishes and irradiated with a single dose of 10 Gy. HL-60 (human promyelocytic leukemia) cells were perfused over the irradiated endothelial cells in a parallel plate flow chamber at shear stress ranging from 0.5 to 2.0 dynes/cm2. Flow cytometry was used to quantify the expression of E-selectin and ICAM-1 on the various endothelial cells. Flow cytomeric analysis revealed an upregulation of ICAM-1 expression on all three cell types postirradiation (post-IR), and an upregulation of E-selectin expression only on HDMEC post-IR. E-selectin expression was detected on control HDMEC, but at a lower level than that detected on post-IR HDMEC. Flow assays revealed a significant increase in the number of rolling and firmly adherent HL-60 cells on post-IR HDMEC at shear stress < or =1.5 dynes/cm2; pretreatment of control and irradiated HDMEC with antibodies to E-selectin and ICAM-1 significantly diminished the number of rolling and firmly adherent HL-60 cells, respectively. No rolling or firm adhesion of HL-60 cells was observed on HUVEC or HMEC-1 monolayers post-IR. These findings suggest that ICAM-1 is upregulated on irradiated HDMEC, HUVEC, and HMEC-1. E-selectin is upregulated to a functional level only on irradiated HDMEC, and not on irradiated HUVEC or HMEC-1.

Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells

The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown–knockin experimental system to stably express either fully functional form or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands.

Molecular mechanism of allogeneic CD8+ T cell-induced apoptosis of vascular endothelial cells

To investigate the molecular mechanism of allogeneic CD8+ T cell-induced apoptosis of vascular endothelial cells. Allogeneic CD8+ T cells were isolated from PBMC by positive selection using magnetic beads coated with anti-CD8 antibody. Apoptosis of human umbilical vein endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HDMEC) were detected by Annexin V-FITC labeling. Gene and protein expression of proteinase-activated receptor-1 (PAR-1) in vascular endothelial cells were tested by RT-PCR and Western blot. Western blotting was also used to detect the change of MAPK and Caspase-3 expression in vascular endothelial cells. The effects of SFLLRN (PAR-1 agonist), ATAP2 (PAR-1 antibody), SB203580 (inhibitor of p38MAPK), SP600125 (inhibitor of JNK) upon apoptosis were also examined. After co-culturing with allogeneic CD8+ T cells for 24 h and 48 h, the apoptotic rates of HUVEC were 51.7% +/- 4.1% and 29.4% +/- 3.3% respectively (P < 0.01, vs untreated HUVEC) and those of HDMECs 28.9% +/- 2.2% and 15.2% +/- 1.8% respectively (P < 0.01, vs untreated HDMEC). The effect of PAR-1 agonist upon apoptosis of HUVEC and HDMEC similar to that of allogeneic CD8+ T cells. These effects were largely prevented by ATAP2 and SB203580 (P < 0.05). Allogeneic CD8+ T cells and PAR-1 agonist enhanced the cleavage of Caspase-3 and led to p38MAPK phosphorylation. Allogeneic CD8+ T cells induced the apoptosis of vascular endothelial cells through PAR-1 dependent modulation of intrinsic apoptotic pathway via the cleavage of Caspase-3 and the phosphorylation of p38MAPK.

Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells

Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.

Antigen Presentation by Human Microvascular Endothelial Cells Triggers ICAM-1-Dependent Transendothelial Protrusion by, and Fractalkine-Dependent Transendothelial Migration of, Effector Memory CD4+ T Cells

TCR engagement on adherent human effector memory CD4(+) T cells by TNF-treated HUVECs under flow induces formation of a transendothelial protrusion (TEP) by the T cell but fails to induce transendothelial migration (TEM). In contrast, TCR engagement of the same T cell populations by TNF-treated human dermal microvascular cells (HDMEC) not only induces TEP formation, but triggers TEM at or near the interendothelial cell junctions via a process in which TEP formation appears to be the first step. Transduction of adhesion molecules in unactivated HDMEC and use of blocking Abs as conducted with TNF-activated HDMEC indicate that ICAM-1 plays a nonredundant role in TCR-driven TEP formation and TEM, and that TCR-driven TEM is also dependent upon fractalkine. TEP formation, dependence on ICAM-1, and dependence on fractalkine distinguish TCR-induced TEM from IP-10-induced TEM. These in vitro observations suggest that presentation of Ag by human microvascular endothelial cells to circulating CD4(+) effector memory T cells may function to initiate recall responses in peripheral tissues.

Activation of Src by inhibition of Csk does not decrease endothelial barrier function

Endothelial barrier function in the vasculature is known to be tightly regulated by the adherens junction (AJ). Recent studies have suggested that Src phosphorylation of AJ proteins contributes to a decrease in endothelial barrier function following cardiac ischemia and stroke. We sought to develop an in vitro model of Src activation in order to determine the mechanism of AJ phosphorylation, particularly p120, in Src-induced loss of barrier function. In these studies we activated endogenous Src by expressing a dominant negative form of c-terminal-src kinase (DN-Csk), a kinase known to inactivate Src by phosphorylation of tyrosine 527. Indeed, expression of DN-Csk in confluent human lung or human dermal microvascular cells activated endogenous Src as shown by increases in the dephosphorylation of tyrosine 527 and phosphorylation of tyrosine 416. In addition expression of DN-Csk resulted in a time dependent increase in global tyrosine phosphorylation including phosphorylation of paxillin, a protein found in cell-matrix AJs. Surprisingly, we found that increased Src activation following expression of DN-Csk is not sufficient to cause a loss of barrier function in endothelial cells. These results suggest that the context of Src activation is critical to modulating endothelial barrier function and suggests that Csk may play a greater role in regulation of cell-matrix junctions, as shown by paxillin phosphorylation, than at cell-cell junctions. K02-HL-04332; R01-HL-68079.

Vascular and Biology 01

Background: Endothelin-1 (ET-1) is a 21 amino acid peptide with vasoconstrictor and mitogenic properties. We observed that ET-1 is expressed around areas of angiogenesis in ischaemic skeletal muscle, and sought to determine whether ET-1 provides an angiogenic stimulus to human microvascular cells. Methods: Human dermal microvascular cells (HMEC-1) were grown on growth factor reduced Matrigel, with or without ET-1 at various concentrations. Synergy with the angiogenic factor vascular endothelial growth factor (VEGF) was sought by co-adding ET-1 with VEGF. In addition, we measured the effects of selective inhibitors of the ET-1 receptor. Angiogenesis was measured after 24-h treatment by visual analysis of microvessel growth and by scanning the mean optical density of images of the angiogenic field. Results: ET-1 enhanced angiogenesis in the presence of VEGF (P < 0.02). The ET(B) receptor antagonist BQ788 suppressed angiogenesis below basal levels (P < 0.04), whereas the ET(A) receptor antagonist had no effect. Microvascular cells were found to release ET-1 when grown on Matrigel, with enhancement following treatment with VEGF. Conclusions: These data suggest that in human microvascular cells, ET-1 is an important autocrine growth factor, acting downstream and in concert with VEGF to stimulate angiogenesis. We suggest that the mitogenic properties of ET-1 are ET(B) mediated, unlike the effects on vascular tone, which are ET(A) mediated. The separation of these effects might provide benefit to patients with peripheral vascular disease, where selective ET(A) receptor antagonists could promote vasodilatation whilst preserving the ET(B) mediated angiogenic effects.

Substance P Activates Coincident NF-AT- and NF-κB-Dependent Adhesion Molecule Gene Expression in Microvascular Endothelial Cells Through Intracellular Calcium Mobilization

AbstractUpon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which modulate responses in the skin by activating a number of target cells via neurokinin receptors. We have demonstrated that SP preferentially binds to the NK-1R on human dermal microvascular cells, resulting in increased intracellular Ca2+ and induction of ICAM-1 and VCAM-1 expression. In the current studies, we identify specific elements in the regulatory regions of ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. SP treatment of human dermal microvascular endothelial cells leads to coincident activation and binding of the transcription factor NF-AT to the −191/−170 region of the ICAM-1 gene (a region bound by activated p65/p65 homodimers in response to TNF-α), and NF-κB (p65/p50) to tandem NF-κB binding sites at −76/−52 of the VCAM-1 gene. The SP-elicited intracellular Ca2+ signal was required for activation and subsequent binding of both NF-AT and NF-κB. The transacting factor induction by SP was specific, since a selective NK-1R antagonist blocked SP activation and subsequent NF-AT and NF-κB activation and binding. These data demonstrate coincident activation of NF-AT and NF-κB via SP-induced intracellular Ca2+ mobilization and indicate a crucial role for neuropeptides in modulating localized cutaneous inflammatory responses.

Histamine-induced Changes in the Actin Cytoskeleton of the Human Microvascular Endothelial Cell line HMEC-1

Increased permeability of the microvascular endothelium is a component of the inflammatory response. Inflammatory mediators such as histamine contribute to this permeability change. Modulation of cytoskeletal F-actin has been implicated as part of the cellular mechanism involved. Permeability changes occur predominantly at the microvascular level while the majority of current knowledge stems from research on cells from large vessels. We have therefore utilized an immortalized human dermal microvascular cell line, HMEC-1. Confluent monolayers were exposed to histamine (10 μ m, 100 μ m) for 1, 5, 10 or 15 minutes. F-Actin changes were detected by labelling with FITC-conjugated phalloidin. Histamine exposure resulted in the rounding of cells with the formation of intercellular gaps. The percentage of rounded cells and the number of gaps increased with exposure time. F-actin was redistributed from a peripheral band in control cultures to a perinuclear zone. Continual presence of the agonist was required for these phenotypic changes to occur. Removal of histamine caused reversal of these observations. Cells exposed to histamine for 1 minute needed 15 minutes to recover their normal morphology and F-actin distribution. These reversible effects suggest that F-actin redistribution maybe part of the microvascular cell response to histamine.

Angiostatin Diminishes Activation of the Mitogen-Activated Protein Kinases ERK-1 and ERK-2 in Human Dermal Microvascular Endothelial Cells

Angiostatin is an endogenous inhibitor of angiogenesis that was isolated from tumor-bearing mice. It has been established that angiostatin inhibits endothelial cell proliferation; however, the underlying mechanisms remain to be elucidated. Here we report that angiostatin reduces transiently the phosphorylation of the mitogen-activated protein kinases ERK-1 and ERK-2 in human dermal microvascular cells, but not in human vascular smooth muscle cells or human dermal fibroblasts. We demonstrate that angiostatin diminishes ERK activation by basic fibroblast growth factor and vascular endothelial growth factor. Dephosphorylation of ERK and other tyrosine-phosphorylated proteins was blocked by pretreatment of the cells with sodium meta-vanadate, an inhibitor of protein tyrosine phosphatases, indicating that angiostatin signaling may require the activity of a tyrosine phosphatase. Concentrations of angiostatin that inhibited ERK activation also inhibited basic fibroblast growth factor-stimulated collagen gel invasion by endothelial cells, but did not affect endothelial cell proliferation. We thus show that angiostatin inhibits primarily the invasion of endothelial cells and exerts minimal (if any) effects on their proliferation. Invasion is a process that involves proteolysis, adhesion and migration, all of which have been linked to ERK signaling.

A Novel Technique for Culture of Human Dermal Microvascular Endothelial Cells under either Serum-Free or Serum-Supplemented Conditions: Isolation by Panning and Stimulation with Vascular Endothelial Growth Factor

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptor kdr,since mRNA for kdrwas detected using RT–PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.

6 Cytokine stimulated human dermal microvascular endothelial cells (HDMEC) are a major source for interleukin 6

6 Cytokine stimulated human dermal microvascular endothelial cells (HDMEC) are a major source for interleukin 6