Skin Endothelial Cells Research Articles

Immunomodulating Properties of Stored Product Mite Extracts on Human Dermal Fibroblasts

RATIONALE: Since dust and/or scabies mite extracts are the sources of bioactive components that influence the function of lung epithelial cells, fibroblasts, keratinocytes, skin endothelial cells, lymphocytes, monocytes, and dendritic cells, we undertook a study to determined if components in storage mite extracts influenced the function of fibroblasts that are a common cell type in many tissues. METHODS: Normal human dermal fibroblasts were cultured and then exposed to whole body extracts from the stored product mites Acarus siro (AS), Chortoglyphus arcuatus (CA), and Lepidoglyphus destructor (LD) in the presence or absence of lipopolysaccharide (LPS). Cytokine secretion by the cells was measured by ELISA. RESULTS: Components in the body extracts of all three species of mites induced the secretion of IL-8 while CA and LD induced secretion of IL-6 and MCP-1 generally in a dose dependent manner. CA extracts induced greater secretion of IL-6 than did LD while LD and CA induced secretion of greater amounts of IL-8 at lower doses than AS. There was no mite-induced secretion of IL-1ra, IL-1α, IL-1β, IL-7, IL-15, MIP-1α, M-CSF, TNFα, or eotaxin. CA body and fecal extracts were fractionated by isoelectric focusing (pH 2.0-12.0). Fractions of pI 5.8-6.3 of the CA body extract induced secretion of IL-6 while no fraction of the fecal extract induced IL-6 secretion. CONCLUSIONS: This study demonstrated that components of storage mite extracts influence the secretion of IL-6, IL-8, and MCP-1 by dermal fibroblasts and therefore may influence the course of an inflammatory and immune reaction.

Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro

The development of vessel-like structures in vitro to mimic as well as to realize the possibility of tissue-engineered small vascular networks presents a major challenge to cell biologists and biotechnologists. We aimed to establish a three-dimensional (3-D) culture system with an endothelial network that does not require artificial substrates or ECM compounds. By using human skin fibroblasts and endothelial cells (ECs) from the human umbilical vein (HUVECs) in diverse spheroid coculture strategies, we verified that fibroblast support and modulate EC migration, viability, and network formation in a 3-D tissue-like stromal environment. In mixed spheroid cultures consisting of human ECs and fibroblasts, a complex 3-D network with EC tubular structures, lumen formation, pinocytotic activity, and tight junction complexes has been identified on the basis of immunohistochemical and transmission electron microscopic imaging. Tubular networks with extensions up to 400 mum were achieved. When EC suspensions were used, EC migration and network formation were critically affected by the status of the fibroblast. However, the absence of EC migration into the center of 14-day, but not 3-day, precultured fibroblast spheroids could not be attributed to loss of F viability. In parallel, it was also confirmed that migrated ECs that entered cluster-like formations became apoptotic, whereas the majority of those forming vessel-like structures remained viable for >8 days. Our protocols allow us to study the nature of tubule formation in a manner more closely related to the in vivo situation as well as to understand the basis for the integration of capillary networks in tissue grafts and develop methods of quantifying the amount of angiogenesis in spheroids using fibroblast and other cells isolated from the same patient, along with ECs.

Sheet‐Based Tissue Engineering: From Bench Top to the First Clinical Use of a Completely Biological Tissue Engineered Blood Vessel

The initial vision of Tissue Engineering was to produce living, autologous and completely biological organs for transplant. However, in the case of blood vessels, previous approaches have relied on permanent synthetic scaffolds to provide the requisite mechanical strength. Sheet-Based Tissue Engineering is the first method that allows the in vitro production of mechanically sounds tissues and organs without the need for exogenous scaffolding. Tissue engineered blood vessels (TEBVs) were produced from skin fibroblasts and endothelial cells isolated from older patients suffering from vascular disease and/or diabetes. In animal models, TEBVs showed long-term patency (over 8 months), no mechanical failure and positive histological remodeling. A small clinical trial was initiated to asses the potential of the TEBV as a arteriovenous shunt. At the time of submission, TEBVs produced from the cells of patients (age: 67±11 y, n=6) have all demonstrated satisfactory burst pressures (3658±1074 mmHg), suture retention strength (192±29 gf) and compliance (2.6±0.4 %/100mmHg). First clinical use revealed that these TEBVs can be easily sutured using standard equipment and technique. Grafts showed high flow (>1L/min), no signs of mechanical failure and patency through 6 months (on going). Grafts resisted repeated puncture and could be use for hemodialysis. IVUS studies revealed an almost 3-fold increase in compliance without vessel dilatation. These data demonstrate the feasibility of producing completely biological and autologous TEBVs for arterial bypass from cells of elderly patients in poor health. Partly funded by NIH/NHLBI R44HL64462.

Self-Organization of Skin Cells in Three-Dimensional Electrospun Polystyrene Scaffolds

Much research in tissue engineering focuses on the synthesis of complex three-dimensional polymer scaffolds containing functional biomolecules to which cells are introduced. Typical scaffolds for skin tissue engineering are macroscopically porous with struts or fibers approximately 10 microm thick at a packing fraction of approximately 0.1. We made a polystyrene scaffold without cell signaling or spatial information by electrospinning and studied the growth of skin fibroblasts, keratinocytes, and endothelial cells, as single and cocultured populations in the presence and absence of fetal calf serum. In the absence of serum, keratinocytes, fibroblasts, and endothelial cells did not grow when cultured alone. However, when fibroblasts were cocultured with keratinocytes and endothelial cells, expansion of keratinocytes and endothelial cells occurred even in the absence of serum. Furthermore, cells displayed native spatial three-dimensional organization when cultured at an air-liquid interface, even when all three cell types were introduced at random to the scaffold. This study shows that coculture with fibroblasts enables keratinocytes and endothelial cells to proliferate without serum, but also to self-organize according to the native epidermal-dermal structure given the symmetry-breaking field of an air-liquid interface.

Low recruitment of immune cells with increased expression of endothelial adhesion molecules in margins of the chronic diabetic foot ulcers

Diabetic foot skin close to an ulcer shows only a few infiltrating cells compared to nondiabetic inflamed tissues. Diabetes is characterized by thickened basement membrane of the blood arterioles and capillaries. This may affect the transcapillary transport of immune humoral factors and cells to the extravascular space. We analyzed by immunohistochemistry the phenotype and expression pattern of adhesion molecules on leukocyte, dermal fibroblast, and endothelial cells in diabetic foot ulcers. Although there was accumulation of granulocytes on the surface and superficial layers of the granulation tissue, rare perivascular granulocyte infiltrates in the dermis were seen. Moreover, lack of macrophage and CD3+ T cell infiltrates was observed. In contrast, there was increased intensity of CD1a staining of Langerhans cells in the epidermis and papillary dermis (p < 0.05). Fibroblasts revealed increased presence in the ulcer margins compared with normal skin (p < 0.05). Skin endothelial cells expressed stronger von Willebrand factor and E-selectin compared with normal skin (p < 0.05). Our study provides evidence that increased expression of endothelial cell adhesion molecules responsible for immunocyte extravasation is not associated with increased inflammatory cell infiltration of the ulcerated diabetic foot tissue. We suggest that the healing process of diabetic foot ulcers may be hampered by mechanisms decreasing accumulation of leukocytes. This implies that pharmacological or biological stimulation of leukocyte extravasation into the ulcer tissue should be tried.

Adhesion properties of adhesion‐regulating molecule 1 protein on endothelial cells

Numerous adhesion molecules have been described, and the molecular mechanisms of lymphocyte trafficking across the endothelium is starting to be elucidated. Identification of the molecules involved in the organoselectivity of this process would help in the targeting of drug therapy to specific tissues. Adhesion-regulating molecule-1 (ARM-1) is an adhesion-regulating molecule previously identified on T cells. It does not belong to any known families of adhesion molecules. In this study, we show the presence of ARM-1 in endothelial cells, the adhesion partners of lymphocytes. ARM-1 mRNA was found to be differentially expressed in endothelial cell lines of various tissue origin and lymphocyte cell lines. Interestingly, ARM-1 is absent from skin endothelial cells. In our assay, skin endothelial cells display a distinct capacity to mediate adhesion of activated T lymphocytes. Overexpression of ARM-1 in skin endothelial cells increased adhesion of CEMT4 and NK lymphocytes, confirming that ARM-1 also regulates adhesion in endothelial cells. We also show that ARM-1 is a cytosolic protein associated with the plasma membrane. However, no cell surface expression of the protein was observed. These results suggest an indirect role of ARM-1 in adhesion rather than a direct role as an adhesion molecule itself.

Varicella-Zoster virus pathogenesis and immunobiology: new concepts emerging from investigations with the SCIDhu mouse model.

These experiments provide evidence in support of a critical role for T-cell tropism in VZV pathogenesis and suggest that the prolonged varicella incubation period represents the time required for VZV to overcome previously unrecognized potent innate antiviral responses, especially IFN-α production, mediated directly by epidermal cells. The initial phase of VZV pathogenesis appears to be facilitated by virus-mediated modulation of MHC-I and MHC-II expression and by the failure of VZV to trigger up-regulation of inflammatory adhesion molecules on capillary endothelial cells in skin. Eliminating ORF47 kinase activity impaired virion production and envelopment, which was associated with a dramatic reduction of VZV virulence in skin and a complete block of VZV infectivity for T cells in SCIDhu xenografts. Our interpretation of these observations is that VZV T-cell tropism is much more dependent on virion assembly than is VZV replication in epidermal cells. Some VZV infectivity is retained in skin if cell fusion functions are preserved, allowing cell-cell spread even when virion production and envelopment are impaired quite significantly. In contrast, VZV T-cell tropism appears to require efficient virion formation and egress for transfer to uninfected cells. Genetically engineered mutations in the VZV genome that result in the retention of some capacity to replicate in skin, at a level sufficient to elicit adaptive immunity to VZV, while eliminating infection of T cells by interfering with virion assembly may yield improved live attenuated VZV vaccines.

Targeted Disruption of MAIL, a Nuclear IκB Protein, Leads to Severe Atopic Dermatitis-like Disease

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IkappaB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor kappaB (IkappaB) zeta. In this study, we generated Mail-/- mice to investigate the roles of MAIL in whole organisms. Mail-/- mice grew normally until 4-8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail-/- mice than in normal. Histopathological analysis indicated that the Mail-/- skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail-/- skin lesions, similar to that observed in the skin of patients with AD. In Mail-/- mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail-/- mouse is a valuable new animal model for research on AD.

Tumor-Associated Endothelial Cells with Cytogenetic Abnormalities

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, although these endothelial cells are structurally and functionally abnormal. However, we hypothesized that given the phenotypic differences between tumor and normal blood vessels, there may be genotypic alterations as well. Mouse endothelial cells were isolated from two different human tumor xenografts, melanoma and liposarcoma, and from two normal endothelial cell counterparts, skin and adipose. Tumor-associated endothelial cells expressed typical endothelial cell markers, such as CD31. They had relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells were cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. Multicolor FISH indicated that the structural chromosomal aberrations in tumor endothelial cells were heterogeneous, indicating that the cytogenetic alterations were not clonal. There was no evidence of human tumor-derived chromosomal material in the mouse tumor endothelial cells. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid, had normal centrosomes, and remained cytogenetically stable in culture even up to 20 passages. FISH analysis of tumor sections also showed endothelial cell aneuploidy. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment.

UVB Radiation-Mediated Expression of Inducible Nitric Oxide Synthase Activity and the Augmenting Role of Co-Induced TNF-α in Human Skin Endothelial Cells

Nitric oxide (NO) plays a pivotal role in ultraviolet radiation-induced inflammation in human skin. We had earlier reported on the inducible nitric oxide synthase (iNOS) inducing activity of UVA radiation. We now demonstrate that UVB-exposure induces expression of the iNOS in vessel endothelia of normal human skin and in cultured human dermal endothelial cells (HUDEC), although by a molecular mechanism different from UVA-mediated induction. With HUDEC, UVB induces iNOS expression and leads to significant enzyme activities, although at app. 5-fold lower levels than can be achieved with proinflammatory cytokines. In contrast to our earlier observation with UVA, cytokine-challenge combined with simultaneous UVB-exposure had no additive effects on iNOS expression nor activity. Interestingly, a time-delay between UVB-irradiation and cytokine-challenge enhances endothelial iNOS enzyme activity 2.5-fold over cytokines activation only. This time-dependent effect strongly correlates with UVB-induced endothelial TNF-alpha expression. In HUDEC addition of TNF-alpha results in enhanced expression of the inducible arginine transporter system CAT-2 essential for substrate supply and thus iNOS activity. In summary, UVB induces iNOS mRNA and enzyme activity in HUDEC. Moreover, UVB augments CAT-2 expression through a TNF-alpha- dependent mechanism which essentially contributes to increased iNOS activity.

Expression of Indoleamine 2,3-Dioxygenase in Dermal Fibroblasts Functions as a Local Immunosuppressive Factor

As a possible way of making a non-rejectable skin substitute, here, we ask the question of whether the expression of indoleamine 2,3-dioxygenase (IDO) selectively suppresses immune, but skin, cell proliferation. To address this question, a series of experiments in which adenovirus (Ad-IDO) infected IDO expressing dermal fibroblasts were co-cultured with different types of immune cells were carried out. The immune cells were then harvested and evaluated for propidium iodide (PI) positive cells by FACS analysis. TUNEL assay was also carried out to determine the apoptotic status of these cells. The results showed that the expression of IDO in dermal fibroblasts significantly induces apoptotic death of PBMC, CD4(+)-, CD8(+)- and B cell-riched primary lymphocytes, Jurkat cells, and THP-1 cells. IDO-mediated damage of immune cells was restored by an addition of tryptophan and IDO inhibitor. Using the same approaches, we also demonstrated that skin cells and endothelial cells are remarkably resistant to tryptophan-deficient environment. Furthermore, no significant difference in cell proliferation between Ad-GFP (control)- and Ad-IDO-GFP-infected either keratinocytes or fibroblasts, was found. The results of this study, therefore, suggest that the expression of IDO by dermal fibroblasts mediates immune cell damage and this may shed a new light toward developing a non-rejectable skin substitute in the future.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

Ultraviolet A1 Radiation Induces Nitric Oxide Synthase-2 Expression in Human Skin Endothelial Cells in the Absence of Proinflammatory Cytokines

Skin exposure to ultraviolet radiation from sunlight causes erythema and edema formation as well as inflammatory responses. As some of these ultraviolet-induced effects are potentially mediated by nitric oxide synthases, we examined the role of cytokines and ultraviolet A1 radiation (340-400 nm) on the expression of the nitric oxide synthase-2 in endothelia of normal human skin biopsies during short-term organ culture as well as expression and activity of the nitric oxide synthase-2 in in vitro cell cultures of human dermal endothelial cells. Both, cytokine challenge (interleukin-1beta + tumor necrosis factor-alpha + interferon-gamma) but also ultraviolet A1 exposure (50 J per cm2) in the absence of cytokines led to the expression of nitric oxide synthase-2 in human skin organ cultures as shown by immunohistochemistry. Moreover, exposing human dermal endothelial cell cultures to proinflammatory cytokines but also to ultraviolet A1 radiation (6-24 J per cm2) in the absence of cytokines resulted in significant nitric oxide synthase-2 mRNA and protein expression as well as enzyme activity. Ultraviolet A1 irradiation of cytokine activated cells led to further increases in nitric oxide synthase-2 mRNA, protein expression, and enzyme activity. Moreover, a reporter gene assay using a human nitric oxide synthase-2 promoter construct provide evidence that ultraviolet A1, in the absence of cytokines, induces nitric oxide synthase-2 expression and activity, as previously shown for cytokines. Thus, the results presented here demonstrate for the first time that in dermal endothelia of human skin ultraviolet A1 radiation alone represents a proinflammatory stimulus sufficient to initiate nitric oxide synthase-2 expression as well as activity comparable with the respective response seen in the presence of proinflammatory cytokines.

Sustainable systemic delivery via a single injection of lentivirus into human skin tissue.

The skin offers a tissue site accessible for delivery of gene-based therapeutics. To develop the capability for sustained systemic polypeptide delivery via cutaneous gene transfer, we generated and injected pseudotyped HIV-1 lentiviral vectors intradermally at a range of doses into human skin grafted on immune-deficient mice. Unlike Moloney murine leukemia virus (MLV)-based retrovectors, which failed to achieve detectable cutaneous gene transfer by this approach, lentivectors effectively targeted all major cell types within human skin tissue, including fibroblasts, endothelial cells, keratinocytes, and macrophages. After a single injection, lentivectors encoding human erythropoietin (EPO) produced dose-dependent increases in serum human EPO levels and hematocrit that increased rapidly within one month and remained stable subsequently. Delivered gene expression was confined locally at the injection site. Excision of engineered skin led to rapid and complete loss of human EPO in the bloodstream, confirming that systemic EPO delivery was entirely due to lentiviral targeting of cells within skin rather than via spread of the injected vector to visceral tissues. These findings indicate that the skin can sustain dosed systemic delivery of therapeutic polypeptides via direct lentivector injection and thus provide an accessible and reversible approach for gene-based delivery to the bloodstream.

Fabry's Disease Mimicking an Undifferentiated Connective Tissue Disease

We describe a patient with Fabry's disease who for many years was seen in other clinics and was thought to have an undifferentiated connective tissue disease or an incomplete form of CREST syndrome. He presented with polyarthralgias; multiple telangiectasia-like lesions in his oral mucosa, hands, and periumbilical area; mild dysphagia; Raynaud's phenomenon; bilateral leg lymphedema; renal insufficiency; and aseptic bone necrosis at both knees. The diagnosis of Fabry's disease was first suspected when ultrastructural studies on his kidney revealed the typical inclusions characteristic of glycosphingolipidosis. Diagnosis of Fabry's disease was later confirmed by finding similar inclusions in skin endothelial cells and demonstrating a low alpha-galactosidase A activity in plasma. Fabry's disease, although rare, must be considered in the differential diagnosis of patients considered to have "atypical undifferentiated connective tissue diseases," even in the absence of classic angiokeratomas.

Mast cells migrate, but do not degranulate, in response to fractalkine, a membrane-bound chemokine expressed constitutively in diverse cells of the skin.

Mast cells (MC) are anatomically located near nerves and blood vessels in skin and the gastrointestinal tract and tend to localize within certain cutaneous tumors such as neurofibromas. However, the molecular mechanisms by which MC home to these sites are not well characterized. Fractalkine (FK) is a membrane-bound CX3C chemokine that displays constitutive expression in dendritic cells as well as in non-hematopoietic tissues including mammalian brain. Here we show that FK is constitutively expressed by skin endothelial cells, dermal dendrocytes and cells within neurofibromas. By reverse transcription-PCR, FK receptor, CX3CR1, is expressed by cultured murine bone marrow-derived MC (BMMC) of both connective tissue and mucosal phenotypes. Non-activated human dermal MC isolated from neonatal foreskin similarly demonstrated CX3CR1 expression. In chemotaxis assays, FK attracted MC with maximal migration occurring between 25 - 125 ng / ml. BMMC were not stimulated to release proinflammatory mediators in the presence of FK as measured by granule-associated beta-hexosaminidase release. Thus, CX3CR1 is expressed by MC and effectively mediates chemotaxis without inducing degranulation. We propose that the constitutive expression of FK on certain cells in the skin may be a factor in the tissue-specific homing of MC.

Mast cells migrate, but do not degranulate, in response to fractalkine, a membrane-bound chemokine expressed constitutively in diverse cells of the skin

Mast cells (MC) are anatomically located near nerves and blood vessels in skin and the gastrointestinal tract and tend to localize within certain cutaneous tumors such as neurofibromas. However, the molecular mechanisms by which MC home to these sites are not well characterized. Fractalkine (FK) is a membrane-bound CX3C chemokine that displays constitutive expression in dendritic cells as well as in non-hematopoietic tissues including mammalian brain. Here we show that FK is constitutively expressed by skin endothelial cells, dermal dendrocytes and cells within neurofibromas. By reverse transcription-PCR, FK receptor, CX3CR1, is expressed by cultured murine bone marrow-derived MC (BMMC) of both connective tissue and mucosal phenotypes. Non-activated human dermal MC isolated from neonatal foreskin similarly demonstrated CX3CR1 expression. In chemotaxis assays, FK attracted MC with maximal migration occurring between 25 - 125 ng / ml. BMMC were not stimulated to release proinflammatory mediators in the presence of FK as measured by granule-associated beta-hexosaminidase release. Thus, CX3CR1 is expressed by MC and effectively mediates chemotaxis without inducing degranulation. We propose that the constitutive expression of FK on certain cells in the skin may be a factor in the tissue-specific homing of MC.

Peripheral Lymph Node Addressins are Expressed on Skin Endothelial Cells

The term "peripheral node addressins" describes a set of several endothelial adhesion molecules, which collectively bind to L-selectin and react with monoclonal antibody MECA-79. They regulate lymphocyte recirculation through peripheral nodes. Their expression is thought to be restricted to a specialized vascular segment within the node, called the high endothelial venule. In certain chronic skin diseases, however, postcapillary venules of the skin may also acquire a high endothelial venule-like morphology. Employing immunohistochemistry on cryostat sections, we found these skin endothelial cells - like peripheral node high endothelial venules - to be reactive with monoclonal antibody MECA-79. Tissue lysates from the same specimens were then analyzed by immunoprecipitation using recombinant human L-selectin Fc-chimeras followed by immunoblotting using monoclonal antibody MECA-79. In contrast to peripheral node endothelium, which mainly expressed peripheral node addressin moieties of molecular sizes 90-110 kDa and 160 kDa, endothelial cells in cutaneous T cell lymphoma skin lesions expressed an additional and not yet defined 220 kDa peripheral node addressin-like molecule. Most surprisingly, even in normal skin specimens, we found a distinct subset of endothelial cells located around hair follicles constitutively expressing 90-110 kDa peripheral node addressin-like moieties. It is intriguing to speculate that - in analogy to the role of peripheral node addressins in peripheral nodes - the induced expression of peripheral node addressins in chronic T cell mediated skin diseases is responsible for a sustained lymphocyte recruitment. The constitutive expression of peripheral node addressins on perifollicular endothelium may serve for a continuous lymphocyte recirculation through normal skin.

Adhesion molecule expression on skin endothelia in atopic dermatitis: Effects of TNF-α and IL-4

Background: Atopic dermatitis (AD) is characterized by skin infiltrates of leukocytes, such as lymphocytes and eosinophils. Objective: To describe the mechanisms determining this inflammatory process, we have analyzed expression of adhesion molecules and their regulation on skin endothelial cells (ECs). Methods: Expression of adhesion molecules on ECs was analyzed by immunohistochemistry by using Ulex europaeus agglutin 1 as a pan-endothelial marker. Results: Vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin were not found in skin of nonatopic individuals, whereas expression of these surface molecules was observed in nonlesional skin of patients with AD and was even more pronounced in lesional skin or after epicutaneous application of aeroallergen. Induction of adhesion molecule expression was examined on both macrovascular ECs from human umbilical cord vein (HUVECs) and human microvascular ECs (HMEC-1) from skin. TNF-α very potently upregulated adhesion molecule expression in vitro on both EC cell types. To verify the in vivo relevance of TNF-α, we performed TNF-α staining in the skin. TNF-α was observed in the dermis of nonatopic skin, both in chymase-containing mast cells and CD68 + macrophages. The increase in the number of TNF-α–containing cells was concomitant with the increase in adhesion molecule expression in the skin of patients with AD. IL-4 is supposed to be important in atopic diseases because of its IgE- and VCAM-1–inducing properties. However, IL-4 addition failed to induce VCAM-1 expression on HMEC-1, although in the same set of experiments, a clear induction of VCAM-1 expression by IL-4 on HUVECs was demonstrated. Flow cytometry revealed the absence of IL-4 receptor α-chains on HMEC-1 and their presence on HUVECs. Immunohistochemistry examination on skin sections showed no binding of the IL-4R α-chain antibodies to ECs. Conclusion: We conclude that adhesion molecule expression is increased in the skin of patients with AD. Most probably, this increased expression is not a (direct) effect of IL-4 on skin endothelium, but other cytokines, such as TNF-α, might be responsible for this increased adhesion molecule expression. Continuous adhesion molecule expression may facilitate T-cell extravasation in a nonantigen–specific manner, thus explaining the presence of increased T-cell numbers in nonlesional skin of patients with AD. (J Allergy Clin Immunol 1998;102:461-8.)

Quercetin Protects Cutaneous Tissue-Associated Cell Types Including Sensory Neurons From Oxidative Stress Induced By Glutathione Depletion: Cooperative Effects of Ascorbic Acid

Oxidation reactions are essential biological reactions necessary for the formation of high-energy compounds used to fuel metabolic processes, but can be injurious to cells when produced in excess. Cutaneous tissue is especially susceptible to damage mediated by reactive oxygen species and low-density lipoprotein oxidation, triggered by dysmetabolic diseases, inflammation, environmental factors, or aging. Here we have examined the ability of the flavonoid quercetin to protect cutaneous tissue-associated cell types from injury induced by oxidative stress, and possible cooperative effects of ascorbic acid. Human skin fibroblasts, keratinocytes, and endothelial cells were cultured in the presence of buthionine sulfoximine (BSO), an irreversible inhibitor of glutathione (GSH) synthesis. Depletion of intracellular levels of GSH leads to an accumulation of cellular peroxides and eventual cell death. Quercetin concentration-dependently (EC50: 30–40 μM) reduced oxidative injury of BSO to all cell types, and was also effective when first added after BSO washout. BSO caused marked decreases in the intracellular level of GSH, which remained depressed in quercetin-protected cells. Ascorbic acid, while by itself not cytoprotective synergized with quercetin, lowered the quercetin EC50 and prolonged the window for cytoprotection. The related flavonoids rutin and dihydroquercetin also decreased BSO-induced injury to dermal fibroblasts, albeit less efficaciously so than quercetin. The cytoprotective effect of rutin, but not that of dihydroquercetin, was enhanced in the presence of ascorbic acid. Further, quercetin rescued sensory ganglion neurons from death provoked by GSH depletion. Direct oxidative injury to this last cell type has not been previously demonstrated. The results show that flavonoids are broadly protective for cutaneous tissue-type cell populations subjected to a chronic intracellular form of oxidative stress. Quercetin in particular, paired with ascorbic acid, may be of therapeutic benefit in protecting neurovasculature structures in skin from oxidative damage.Copyright © 1997 Elsevier Science Inc.