Tetracycline suppresses ATP gamma S-induced CXCL8 and CXCL1 production by the human dermal microvascular endothelial cell-1 (HMEC-1) cell line and primary human dermal microvascular endothelial cells

Abstract:  Tetracyclines (TCN) have powerful anti-inflammatory properties in addition to their anti-microbial effects. These anti-inflammatory effects are thought to play a role in inhibiting cutaneous inflammation in patients with rosacea and acne; however, the mechanism(s) of this action remains poorly understood. We have previously shown that adenosine-5'-triphosphate (ATP)γS, a hydrolysis-resistant ATP analogue, augments secretion of pro-inflammatory messengers by a human dermal microvascular endothelial cell line (HMEC-1). ATP released by the sympathetic nerves during stress may stimulate release of pro-inflammatory chemokines by dermal vessel endothelial cells, resulting in recruitment of inflammatory cells and exacerbation of inflammatory skin disease. Here we demonstrate that TCN inhibits ATPγS-induced release of pro-inflammatory mediators by HMEC-1 cells and primary human dermal microvascular endothelial cells. TCN dose-dependently inhibited ATPγS-induced augmentation of CXCL8 (interleukin-8) and CXCL1 (growth-regulated oncogene-α) production by HMEC-1 cells and primary human dermal endothelial cells in vitro . TCN and ATPγS did not affect HMEC-1 cell viability as determined by trypan-blue exclusion and cell counts. Inhibition of production of inflammatory mediators by endothelial cells may be one mechanism by which TCN improves inflammatory skin diseases. The ability to inhibit release of inflammatory mediators induced in HMEC-1 cells by purinergic agonists may be a useful way to screen for potential therapeutic agents for cutaneous inflammation.     

Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells

Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell‐based co‐cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF‐1, two well‐known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.     

Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cells

Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3-10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14-15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging.     

The effect of moesin overexpression on ageing of human dermal microvascular endothelial cells

Abstract:  Senescence of microvascular endothelial cells is known to play an important role in the pathophysiology of vascular diseases related to ageing, but the accurate mechanism or related genes are not known. Moesin, a cytoskeletal protein and the most potent candidate as an ageing-related protein, showed obvious changes in expression when compared before and after ageing. In this study, a lentivirus was used to overexpress moesin in endothelial cells. The expression of cell cycle mediators such as p16, cyclin D1 and cdk4, which can be the markers of ageing, was compared by RNA and was shown to be suppressed in moesin overexpressed endothelial cells. In conclusion, it can be said that the expression of moesin delays senescence of human dermal microvascular endothelial cells and this fundamental discovery can be used as a basis for understanding the mechanism of ageing and age-related diseases.     

UV radiation induces the release of angiopoietin-2 from dermal microvascular endothelial cells

In human skin, ultraviolet radiation (UVR)-induced erythema is characterized by the inflammatory and angiogenic activation of dermal endothelial cells. Recently, it has been shown that the release of angiopoietin-2 (Ang-2) from cytoplasmic storages of activated endothelial cells is crucial for the induction of inflammation and angiogenesis. Therefore, we hypothesized that UVR exposure induces the release of Ang-2 from endothelial cells controlling the early steps of erythema formation. In an in vivo study, suction blister fluids generated from UV-irradiated skin showed significantly increased concentrations of Ang-2, vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNFα). Likewise, in vitro UVR exposure of human dermal microvascular endothelial cells (HDMECs) triggered the release of Ang-2 that enhanced the pro-inflammatory response of these cells and facilitated their detachment from smooth muscle cells as evidenced by employing a three-dimensional co-culture spheroid model. These effects were inhibited by angiopoietin-1 (Ang-1), which competes with Ang-2 for binding the endothelial cell Tie2 receptor. Collectively, these observations suggest that UVR triggers the release of endothelial Ang-2 which may promote the destabilization and pro-inflammatory phenotype of the microvascular endothelium. As Ang-1 counteracts UVR-induced effects, stimulating the Ang-1 activity may represent a strategy to stabilize the dermal microcirculatory system, thus protecting against UVR-induced skin damages.     

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells

Skin is one of the most commonly studied tissues for microcirculation research owing to its close correlation of cutaneous vascular function, ageing and age‐related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and ageing in the vascular environment of the skin, we performed a proteomic analysis of plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Among identified proteins, paraoxonase 1 (PON1) was selected in this study. To elucidate the role of PON1 on skin ageing and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked‐down by small interfering RNA (siRNA) targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence‐associated β‐galactosidase stained cells and enlarged and flattened cell morphology. At 48 h post‐transfection, the protein expression of p16 in PON1 siRNA‐treated HDMECs was higher than that in scrambled siRNA‐treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age‐related candidate biomarkers, were decreased by PON1 knock‐down in HDMECs. In conclusion, these results suggest that PON1 functions as an ageing‐related protein and plays an important role in the cellular senescence of HDMECs.     

梅毒螺旋体对人脑微血管内皮细胞趋化因子配体6、8、10表达的影响

目的 探讨梅毒螺旋体对人脑微血管内皮细胞（HBMEC）趋化因子配体（CXCL）表达的影响。方法 将培养的HBMEC分为4组,分别加入用细胞培养液稀释的有活性的梅毒螺旋体、灭活的梅毒螺旋体、脂多糖和细胞培养液（梅毒螺旋体组、灭活梅毒螺旋体组、脂多糖组和空白对照组）刺激6、12、24 h,用荧光定量PCR和酶联免疫吸附试验分别检测细胞中CXCL6、CXCL8和CXCL10基因和蛋白表达水平。利用Transwell小室细胞迁移实验检测梅毒螺旋体刺激后HBMEC对人早幼粒细胞白血病细胞HL-60的趋化作用。结果 在各时间点,梅毒螺旋体组HBMEC中CXCL6、CXCL8和CXCL10的mRNA表达水平均显著高于空白对照组和灭活梅毒螺旋体组（P＜0.05）,而灭活梅毒螺旋体组与空白对照组之间差异无统计学意义（均P＞0.05）。和脂多糖组相比,梅毒螺旋体组的CXCL6和CXCL8 mRNA表达降低（P＜0.05）,CXCL10差异无统计学意义（P＞0.05）。在各时间点,梅毒螺旋体组HBMEC培养上清液中CXCL6和CXCL8含量均高于空白对照组和灭活梅毒螺旋体组（均P＜0.05）,而后2组间差异无统计学意义（均P＞0.05）。各时间点培养上清液中CXCL10含量在梅毒螺旋体组、灭活梅毒螺旋体组和空白对照组间差异无统计学意义（均P＞0.05）;梅毒螺旋体组Transwell小室下室中迁移的HL-60细胞数量（A570）均高于另2组（均P＜0.05）。结论 有活性的梅毒螺旋体可上调HBMEC中CXCL6、CXCL8和CXCL10基因表达水平,增加CXCL6和CXCL8的分泌,增强HBMEC对HL-60细胞的趋化作用,这可能在神经梅毒的发病中起一定作用。     

甘草提取物对人毛乳头细胞增殖和分泌血管内皮生长因子的影响

目的：观察甘草提取物对体外培养的人毛乳头细胞增殖和分泌血管内皮生长因子（VEGF）的影响。方法：采用不同浓度的甘草提取物作用于体外培养的人毛乳头细胞72h,用四甲基偶氮唑蓝（MTT）法测定细胞增殖活性;ELISA法检测细胞培养上清液中VEGF蛋白含量,反转录（RT）-PCR半定量检测细胞中VEGF mRNA表达。结果：与对照组相比,甘草提取物对毛乳头细胞增殖无明显影响;加药组浓度为2．5、5．0、10．0、15．0μg／mL时,甘草提取物促进细胞分泌VEGF,增加细胞内VEGF mRNA表达,差异有统计学意义。结论：甘草提取物对毛乳头细胞增殖无明显影响,但能显著促进毛乳头细胞分泌VEGF,甘草提取物可能是通过增加毛乳头细胞分泌VEGF来促进毛发生长的。     

拉坦前列素对人毛乳头细胞增殖及分泌血管内皮细胞生长因子的影响

目的:观察拉坦前列素对培养的人毛乳头细胞增殖及其分泌血管内皮细胞生长因子(VEGF)的影响。方法:选择0.16、0.80ng/mL和0.004、0.020、0.100μg/mL5个浓度的拉坦前列素作用于体外培养的人毛乳头细胞72h,采用四甲基偶氮唑蓝(MTT)法测定细胞增殖活性,ELISA法检测细胞分泌的VEGF水平。结果:与对照组相比,拉坦前列素对毛乳头细胞增殖无明显影响;加药组药物浓度达0.80ng/mL时细胞分泌的VEGF水平明显升高,差异具有显著性,且升高水平与药物浓度之间呈剂量依赖关系。结论:拉坦前列素对毛乳头细胞增殖无明显影响,但能显著促进毛乳头细胞分泌VEGF,可能是通过VEGF促进毛发生长。     

Leukotriene B4 enhances adherence of human polymorphonuclear leukocytes to dermal microvascular endothelial cells in vitro

Summary Adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells (ECs) is a crucial step in the diapedesis of inflammatory cells to the site of inflammation. We have demonstrated that leukotriene B₄ (LTB₄), a metabolite of the arachidonic acid cascade, and N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) significantly enhance the binding of human PMNs to selected populations of human dermal microvascular endothelial cells (MECs) in vitro. MECs were isolated from the vascular-rich portion of foreskins of newborns. MECs were grown in Iscove's modified Dulbecco's media with 2% prepartum serum and 8% newborn calf serum on 1% gelatin-coated plastic dishes. PMNs isolated from five human donors were added to the culture dishes for varying time intervals (usually 30 min) in the presence and absence of the chemotactic stimuli LTB₄ and FMLP. Addition of PMNs to MECs in the absence of chemotactic stimuli results in baseline binding to the MEC monolayer. About one in every 150 ECs binds more than five PMNs. These selected ECs are randomly distributed throughout the monolayer. LTB₄ from 10^-10 to 10^-7 M increases the number of MECs which selectively bind PMNs by 260% at 10^-7 M. FMLP also increases adherence in qualitatively and quantitatively similar fashion. These data support a role for LTB₄ in the mediation of adherence of neutrophils to dermal MECs. In contrast to other endothelial cells from the large blood vessels, such as from umbilical veins or calf thoracic aortae, PMNs bind only to selected MECs in culture, even when stimulated with LTB₄ or FMLP. These findings suggest a specific subpopulation of MECs which can be induced to express specific finding sites by LTB₄.Part of this work has been presented at the Meeting of the Western Society of Investigative Dermatology in Carmel, February 3–6, 1986     

Comparison of changes in endothelial adhesion molecule expression following UVB irradiation of skin and a human dermal microvascular cell line (HMEC-1)

We have assessed the pattern of dermal endothelial adhesion molecule expression following broadband UVB irradiation in vivo and in vitro. Skin biopsies were taken from 4 human volunteers at baseline and at 4, 8 and 24 h post-irradiation with 2.5 minimal erythema doses of UVB. Sections were stained immunohistochemically for E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD31 and neutrophil elastase. The effect of direct UVB irradiation on E-selectin, ICAM-1 and VCAM-1 was examined in a human dermal microvascular endothelial cell line, HMEC-1. Cultured HMEC-1 were irradiated with 2.5–40 mJ/cm² of UVB, and assessed for adhesion molecule expression by immunofluorescence microscopy and fluorescence-activated cell sorter analysis. In vivo, E-selectin was minimally expressed on EC at baseline and was induced by 4 h following irradiation, P<0.01. ICAM-1 was moderately expressed at baseline and appeared mildly induced at 24 h, although this did not reach statistical significance. VCAM-1 was weakly expressed in unirradiated skin while CD31 was moderately expressed, but neither was induced by UVB irradiation. A significant neutrophilic infiltrate appeared by 8 h and was maximal at 24 h, P<0.05. Neutrophil infiltration correlated with E-selectin expression, r=0.96. In HMEC-1, ICAM-1 was upregulated at 24 h post-irradiation, with an increase in mean channel fluorescence from 100% at baseline to 145 (SD12)%> at 24 h, P<0.05. No change was seen in expression of E-selectin, VCAM-1 or CD31. These studies support the involvement of endothelial adhesion molecules E-selectin and ICAM-1 in UVB-induced inflammation. Whereas ICAM-1 is upregulated by direct irradiation of endothelial cells, E-selectin stimulation appears to be an indirect effect.     

Ultraviolet-A radiation induces adhesion molecule expression on human dermal microvascular endothelial cells

Ultraviolet radiation is capable of inducing numerous skin reactions. Considerable amounts of UVA radiation penetrate the epidermis and reach the microvascular endothelium of the papillary dermis. In order to investigate putative direct effects of UV radiation on endothelial cells, we studied adhesion molecule expression by immunostaining procedures and FACS analysis, following irradiation of normal human skin and cultured human dermal endothelial cells. Enhanced immunostaining for ICAM-1 and E-selectin was detected in biopsies taken after in vivo UVA and UVB irradiation, compared with non-irradiated control skin. On cultured human dermal endothelial cells, however, ICAM-1 and E-selectin were inducible by UVA but not UVB. The induction was dose-dependent, peaking at 20 J/cm² for both adhesion molecules, and time-dependent, peaking after 6 and 24 h for E-selectin and ICAM-1, respectively. Expression of VCAM-1 and PECAM/EndoCAM/CD31 was unaffected by any UV-radiation modality. The functional integrity of irradiated cells was monitored by an exclusion assay of the fluorescent dye 7-AAD, and by staining for the cytoskeletal proteins actin and vimentin. Our results demonstrate that dermal microvascular endothelial cells are a critical and direct target of UVA, and suggest they may play a pivotal role in UV-induced inflammatory skin conditions.     

Overexpression of laminin-8 in human dermal microvascular endothelial cells promotes angiogenesis-related functions

This study examined the effects of endogenous overexpression of laminin-8 on angiogenesis and wound healing in primary human dermal microvascular endothelial cells (HDMECs). HDMECs expressed laminin-8 and laminin-10, but no other laminins, as determined by radioimmunoprecipitation assay using a panel of antibodies to individual laminin chains. To study laminin-8 function, full-length human laminin alpha4 cDNA was retrovirally transferred to HDMEC, and specific overexpression of laminin-8 was verified by Western blot. Laminin-8 overexpression promoted endothelial cell spreading and migration in scratch assays and accelerated angiogenic tubule formation in collagen gel overlay assays. Strong inhibitory effect of beta1 integrin and weak inhibition by alphavbeta3 integrin antibodies were observed in laminin-8-stimulated cell migration, but only beta1 integrin antibodies affected tubule formation. These studies suggest that laminin-8 overexpression may prove to be a useful method to engineer HDMECs to promote angiogenesis and wound repair.     

Expression and modulation of the vitronectin receptor on human dermal microvascular endothelial cells.

Microvascular endothelial cells express a variety of cell-surface integrins in vivo and in vitro with varying affinities for matrix proteins. The vitronectin receptor (VnR), a complex of the alpha v and beta 3 integrin chains, is capable of binding to a variety of matrix proteins that are deposited in injured tissues, including vitronectin, fibrinogen, and thrombin. Staining of frozen sections of human skin with antibodies recognizing the VnR and examination by immunofluorescence microscopy demonstrates staining in a vascular pattern suggesting in vivo expression of the vitronectin receptor on endothelial cells. Examination of pure cultures of human dermal microvascular endothelial cells (HDMEC) by flow-cytometric analysis and enzyme-linked immunosorbent assay confirmed that HDMEC also express cell surface VnR complex in vitro. Stimulation of human dermal microvascular endothelial cells in vitro with agents that stimulate protein kinase C resulted in dose- and time-dependent increases in expression of alpha v and beta 3 integrin chains. Additionally, stimulation with basic fibroblast growth factor induced similar increases, but stimulation with transforming growth factor-beta or interleukin-1 alpha failed to increase VnR expression. Increases in cell-surface VnR expression also correlated with an increased ability of microvascular endothelial cells to bind to vitronectin, but not fibronectin-coated surfaces. Although increases in cell-surface expression of beta 3 paralleled increases in expression of cell-surface alpha v, regulation of mRNA expression was distinct for each chain. These data suggests that microvascular endothelial cells express the VnR complex in vivo, that the cell-surface expression of this integrin on dermal microvascular endothelial cells can be regulated, and that this regulation may be important in cell adherence, cell migration, and wound healing.