J Huazhong Univ Sci Technol［Med Sci］ 32(5):760-766,2012 DOI 10.1007/s11596-012-1031-5 J Huazhong Univ Sci Technol［Med Sci］ 32(5):2012
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Pingyangmycin-regulated Expressions of Adhesion Molecules in Human Venous Malformation Endothelial Cells* Yulin JIA (贾玉林)1, Jun JIA (贾 俊)2, Yifang ZHAO (赵怡芳)2# 1 Department of Oral and Maxillofacial Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2 Department of Oral and Maxillofacial Surgery, College & Hospital of Stomatology, Wuhan University, Wuhan 430070, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012
Summary: Pingyangmycin (bleomycin A5 hydrochloride, PYM) is one of the anti-neoplastic agents which have been commonly used to treat venous malformations. However, the underlying mechanism by which PYM treats venous malformations remains poorly understood. It was reported that venous endothelial cells could recruit neutrophils via adhesion molecules (E-selectin, ICAM-1, ICAM-3, VCAM-1) during the acute/chronic inflammation and subsequent histological fibrosis after sclerotherapy with PYM. This study explored if the expression of E-selectin, ICAM-1, ICAM-3 and VCAM-1 in human venous malformation endothelial cells could be affected by PYM. HVMECs were cultured from human venous malformation tissue. Expressions of E-selectin, ICAM-1, ICAM-3 and VCAM-1 on HVMECs in response to PYM were analyzed by cell ELISA. The relative levels of mRNA expression in the cells were semi-quantified. The results showed that PYM up-regulated the expressions of E-selectin, ICAM-3, VCAM-1 and ICAM-1 in both time- and concentration-dependent manner. Our findings suggested that PYM could induce the expression of adhesion molecules in HVMECs, which might be a possible mechanism by which sclerotherapy by intralesional injection of PYM treats venous malformations. Key words: E-selectin; intercellular adhesion molecule-1; intercellular adhesion molecule-3; vascular cell adhesion molecule-1; cell culture
Venous malformations are common vascular anomalies with a propensity for the head and neck. Treatment alternatives include surgical resection, laser therapy, embolization, sclerotherapy and others. Sclerotherapy is an effective approach for the treatment of venous malformations[1–4]. Bleomycin sclerotherapy can be used for the treatment of congenital lymphatic and vascular malformations of head and neck[5–7]. The mechanism is believed to lie in its hardening effect on the endothelial cells through non-specific inflammatory reaction. But the effect of bleomycin on primary cultured venous malformation endothelial cells in vitro remains unclear. Bleomycin was reported to induce pulmonary inflammation and subsequent fibrosis[8]. Endothelial cells play an important role in pulmonary fibrosis by producing cytokines and adhesion molecules during the inflammatory process[8]. Recruitment of neutrophils through vascular endothelia is crucial for inflammatory process in pulmonary fibrosis. Adhesion of neutrophils to vascular endothelia is a multi-step process involving several cell adhesion molecules. Increased expression of Yulin JIA, E-mail:
[email protected] # Corresponding author, E-mail:
[email protected] * This project was supported by the Natural Science Foundation of Hubei province, China (No. 2010CDB07907).
adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1, on vascular endothelia is a crucial step in the neutrophil adhesion. Several studies demonstrated that the expression of adhesion molecules in endothelial cells was increased in animal models of pulmonary fibrosis models induced with bleomycin[9–12]. Pingyangmycin (bleomycin A5 hydrochloride, PYM) is one of the antineoplastic agents isolated from Streptomyces Pingyangensisn. S. P., which is structurally similar to bleomycin A5. Sclerotherapy with intralesional injection of PYM (Hebei pharmaceutics Co. Ltd., China) has been commonly used in China to treat venous malformations with desirable therapeutic effects achieved. A number of researches concerning sclerotherapy for oral and facial venous malformations by intralesional injection of PYM was reported[13, 14]. PYM can cause injury and detachment of endothelial cells, thicken the vessel wall, leading to lumen narrowing or occlusion, and infiltration of inflammatory cells[15]. Histopathological examination revealed that fibrosis is a common histological change after sclerotherapy with PYM. The target cells, such as endothelial cells, play an important role in the production of pro-fibrotic cytokines, such as IL-8, TGF-β, and adhesion molecules that are involved in the recruitment of neutrophils during the acute and chronic inflammation and subsequent fibrosis. The mechanism of sclerotherapy with PYM has not yet been well clarified, and the ex-
J Huazhong Univ Sci Technol［Med Sci］ 32(5):2012
pressions of adhesion molecules in HVMECs after exposure to PYM have not been reported. This study examined the effect of PYM on the expression of adhesion molecules in HVMECs, with an attempt to understand the mechanism by which PYM treats venous malformations. 1 MATERIALS AND METHODS 1.1 Cell Culture HVMECs were isolated from human venous malformation tissue as described previously[16]. HVMECs used in this experiment were obtained, by surgical resection, from venous malformation of the left buccal mucosa. The study was approved by an institutional review committee. During the surgical operation, a specimen of the venous malformation was dissected and rolled onto piece of sterile gauze to remove as much adherent blood as possible and put into a container containg gentamicin and amphotericin diluted with D-Hanks and was processed immediately for cell culture. A piece of excised tissue specimen was subjected to routine pathological examination for pathological diagnosis of venous malformation. The surgical explants were cut into pieces of 1 mm2 and plated onto 25 cm2 cell culture flask treated with non-pyrogenic polystyrene (Corning, USA). Dulbecco’s modified eagle medium (DMEM, 1 mL) containing L-glutamine (Gibco, USA) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Gibco, USA), 10 U/mL preservative-free heparin, 15 µg/mL EC growth factor (Sigma, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (both from Gibco, USA) was then added. Cultures were maintained in a humidified incubator at 37°C and 5% CO2. After 6 days of culture, the endothelial cells growing out from explants were selected in several steps. First, cell colonies, which did not had the gross morphology of endothelial cells (under an inverted phase-contrast microscope), were detached, by scraping, and then removed from the dish by aspiration. The colonies that exhibited the characteristic cobble-stone morphology of endothelial cells were allowed to proliferate in the dish for another 1 week, selected as aforementioned, and then sub-cultured. Cells generated from these colonies were called HVMECs. The cells were confirmed by their binding of the EC-specific monoclonal antibody. CD34 and Factor VIII-related antigen were utilized as an endothelial marker. The adhesion molecules expression was measured on 2-8 passages of HVMECs. 1.2 Cell Surface Enzyme Linked Immunosorbent Assay (ELISA) Expressions of E-selectin, ICAM-1, ICAM-3 and VCAM-1 on HVMECs in response to PYM were analyzed by direct cell ELISA in 96-well plates as previously described[17]. HVMECs were seeded at a density of 1×104 cell/well into attachment factor-coated 96-well flat-bottomed tissue culture plates (Costar, USA) and exposed to different concentrations of PYM (0, 10, 100, 500 ng/mL, 1 and 10 µg/mL, with 0 serving as control) for different lengths of time (6, 12, 18, 24, 48, 72 h for analyzing the expression of ICAM-1 and ICAM-3 and 2, 6, 8,12, 24, 48 h for E-selectin and VCAM-1) and each
761 test was repeated three times. After appropriate exposure to PYM, HVMECs in 96-well plates were rinsed with 200 µL cold phosphate buffer saline (PBS) and fixed for 15 min in 96% ethanol at 4°C. Subsequently, HVMECs were air-dried for 30 min and stored at 4°C for ≤14 days. HVMECs in 96-well plates were thawed at room temperature and washed with 200 µL PBS, and then incubated with 50 µL of 2% bovine serum albumin (BSA) in PBS for 1 h to block non-specific binding sites. Cells were then incubated with 40 µL/well of the anti-E-selectin mAb (mouse-anti-human antibody class immunoglobulin was purchased from R&D Systems, USA) (1:150) in PBS for 1 h under constant shaking at room temperature. Negative control was incubated without primary mAb under the same condition, and washed three times with fresh PBS. The secondary rabbit anti-mouse Ig and the final horseradish peroxidase-conjugated sheep anti-rabbit antibody were applied in the same manner. Cells were washed again three times and were incubated with 100 µL/well of peroxidase substrate solution (1 µg/mL o-phenylenediamine in citrate buffer, pH=5, containing 0.015% H2O2) and developed in dark under shaking at 37°C for 15 min. Concentrated sulfuric acid (100 µL/well, 20% v/v) was added to stop the reaction. Absorbance at 490 nm was measured by using an ultra micro-plate reader (Bio-Tek Instruments Inc, USA). The microplate was subsequently washed in tap water before 50 µL/well of 0.08% crystal violet in PBS was added and then incubated for 5 min. After a thorough washing in tap water, 100 µL of 33% acetic acid was used to solubilize the nuclear stain and A value, representing the actual cell count per well, was measured at 570 nm. Background was calculated from parallel wells in which the primary mAbs had been replaced by isotype-matched controls. The cellular ELISA signal was considered positive when A value at 490 nm was at least twice the background. ICAM-1, ICAM-3 and VCAM-1 expressions of HVMECs were analyzed in the same manner. The anti-ICAM-1 mAb, anti-ICAM-3 mAb and anti-VCAM-1 mAb (mouse-anti-human antibody class immunoglobulin) were purchased from R&D Systems, USA. Each test was repeated at least three times. 1.3 RT-PCR Confluent 2–8 passage HVMECs were removed from culture flasks with 0.25% trypsin and re-suspended in DMEM containing 20% FBS at 2.5×105 cell/mL. HVMECs were seeded into 6-well cell culture cluster flat-bottomed with Lid Tissue culture plate treated with non-pyrogenic polystyrene (Costar, USA). Cultures were maintained in a humidified incubator at 37°C and in 5% CO2 for 24 h. Endothelial cells were stimulated with different concentrations of PYM (0, 10, 100, 500 ng/mL, 1 and 10 µg/mL, with 0 serving as control) for different time intervals (6, 12,18, 24, 48, 72 h for detecting the expression of ICAM-1 and ICAM-3 and 2, 6, 8, 12, 24, 48 h for E-selectin and VCAM-1) at 37°C in 5% CO2. Total RNA was isolated from cultured HVMECs stimulated with PYM using TRIzol reagent (Gibco, USA). Purified RNA was re-suspended in 20 µL RNase-free water and quantified in terms of absorbance at 260 nm. The purity was assessed by OD260/OD280 ratio by employing UV-vis recording spectrophotometer (SHIMADZU, Japan) and the samples were stored at –70°C for
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J Huazhong Univ Sci Technol［Med Sci］ 32(5):2012
later RT-PCR. Takara RNA PCR kit, Japan, was used to measure the adhesion molecules mRNA expression of PYM-stimulated HVMECs. Reverse transcription of total RNA into complementary DNA (cDNA) was performed by incubation with 0.5 µL avian myeloblastosis virus reverse transcriptase XL (5 U/µL), 1 µL dNTP (10 mmol/L), 2 µL MgCl2 (25 mmol/L), 2 µL 5×RT buffer, 0.5 µL Oligo-dT-adaptor primer (2.5 pmol/µL), 0.25 µL RNase inhibitor (40 U/µL), 1.75 µL RNase-free dH2O, and 2 µL total RNA (
