Study of novel coating strategy for coronary stents: simutaneous coating of VEGF and anti- CD34 antibody / Estudo da nova estratégia de revestimento para stents coronários: revestimento simultâneo de VEGF e anticorpo anti-CD34

Abstract Introduction: Intravascular coronary stenting has been used in the treatment of coronary artery disease (CAD), with a major limitation of in-stent restenosis (ISR). The 316 stainless steel has been widely used for coronary stents. In this study, we developed a novel coating method to reduce ISR by simultaneously coating vascular endothelial growth factor (VEGF) and anti-CD34 antibody on 316L stainless steel. Methods: Round 316L stainless steel sheets in the D-H group were polymerized with compounds generated from condensation reaction of dopamine and heparin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Sixteen sheets from the D-H group were further immersed into 1ug/ml VEGF165 and 3mg/ml heparin sodium one after another for 10 times, and named as the D-(H-V)10 group. Eight sheets from the D-(H-V)10 group were coated with anti-CD34 antibody and termed as the D-(H-V)10-A group. Immunofluorescence assay and ELISA were used to evaluate whether the 316L stainless steel disks were successfully coated with VEGF and anti-CD34 antibody. Results: The results of immunofluorescence assay and ELISA showed that VEGF could be detected in the D-(H-V)10 and D-(H-V)10-A group, suggesting the steel sheets were successfully covered with VEGF. Anti-CD34 antibody could only be observed in the D-(H-V)10-A group, which was the only group coated with CD34 antibody. Both results suggested that the 316L stainless steel sheets were successfully coated with VEGF and anti-CD34 antibody. Conclusion: Our study developed a method to simultaneously coat VEGF and anti-CD34 antibody to stainless metal steel. This research serves as a fundamental role for a novel coating strategy. .

Resumo Introdução: O stent coronário intravascular tem sido utilizado no tratamento de doença arterial coronária, com uma maior limitação de restenose intra-stent (RIS). O aço inoxidável 316 tem sido amplamente utilizado para stents. Neste estudo, foi desenvolvido um novo método de revestimento para reduzir a RIS para revestir simultaneamente o fator de crescimento endotelial vascular (VEGF) e anti-CD34 em aço inoxidável 316L. Métodos: Placas de aço inoxidável 316L redondas no grupo DH foram polimerizadas com compostos gerados a partir da reacção de condensação de dopamina e heparina utilizando N- (3-dimetilaminopropil) -N'-etilcarbodiimida (EDC) e N-hidroxissuccinimida (NHS). Dezesseis folhas a partir do grupo DH foram ainda imersas em 1 ug/ml de VEGF 165 e 3 mg/ml de heparina sódica, um após outro por 10 vezes, sendo denominado como o grupo D-(HV)10. Oito folhas de D-(HV)10 foram revestidas com anticorpo anti-CD34 e denominado como grupo D-(HV)10-A. Testes de imunofluorescência e ELISA foram usados para avaliar se os discos de aço inoxidável 316L foram revestidos com sucesso com VEGF e anticorpo anti-CD34. Resultados: Os resultados dos testes de imunofluorescência e ELISA mostraram que o VEGF pôde ser detectado nos grupos D-(HV)10 e D-(HV)10-A, evidenciando que as chapas de aço foram cobertas com VEGF com sucesso. O anticorpo anti-CD34 podia apenas ser observado no grupo D-(HV)10-A, o único grupo revestido com anticorpo CD34. Ambos os resultados sugerem que as chapas de aço inoxidável 316L foram revestidas com sucesso com VEGF e anticorpo anti-CD34. Conclusão: Nosso estudo desenvolveu um método para revestir simultaneamente VEGF e anti-CD34 de aço inoxidável. Esta pesquisa tem um papel fundamental para a nova estratégia de revestimento. .

Effects of Platelet Lysate Preparations on the Proliferation of HaCaT Cells

BACKGROUND: Standard protocols are lacking for the preparation of platelet lysates (PL) as an alternative to using fetal bovine serum as a cell culture supplement. This study aimed to establish optimum conditions for preparing PL for use in cell cultures. METHODS: Cell density in three pooled platelet concentrates (PC) were adjusted to 1x10(12)/L and 2x10(12)/L. PL was prepared from PC by 1 to 3 freeze-thaw (FT) cycles. HaCaT cells were cultured in media supplemented with 5% or 10% PL. Cell numbers were estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Growth factors were quantified by using the Luminex 200 system (Luminex Corporation, USA). RESULTS: Cell proliferation rates in the presence of PLs were similar when prepared from PCs of both cell densities. The rates were higher in media containing 5% PL than 10% PL when prepared by two FT cycles. Concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB/BB (PDGF-AB/BB), PDGF-AA, and epidermal growth factor (EGF) were significantly higher in PL prepared from PC with a cell density of 2x10(12)/L than 1x10(12)/L PC. However, only VEGF and PDGF-AA concentrations in PLs were correlated with HaCaT cell counts. CONCLUSIONS: The 5% PL from PC with a cell density of 1x10(12)/L prepared by two FT cycles treatment was the most effective condition that supported steady HaCaT cell proliferation. Our finding may be useful for preparing PL-supplemented cell culture media.

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.

Immunohistochemical expression of cyclooxy genase- 2 [COX - 2] and vascular endothelial growth factor [VEGF] in oral: premalignant lesions and squamous cell carcinoma

Squamous cell carcinoma [SCC] represents more than 90% of all head and neck cancers. It can arise de novo or from premalignant lesions. Seventy to ninety percent of all precancerous oral lesions have the potential to develop into malignant phenotype. Early detection and identification of the molecular changes that are responsible for tumor development and progression is of paramount importance in medical and surgical intervention with improved outcome. Angiogenesis is an essential condition for the development and proliferation of malignant tumors. Cyclooxygenase [Cox-2] as well as vascular endothelial growth factor [VEGF] are two epitopes among the different pro angiogenic molecules that have been investigated in literature in relation to tumor angiogenesis. The present study aimed at delineation of the molecular expression profile of Cox-2 and VEGF factors in oral cavity premalignant dysplatic lesions and SCC and its relation to tumor development, grade and stage of the tumor. Thirty cases were studied, which included 12 tongue lesions, 3 in the cheek and 15 in the palate and mandible. The studied cases were categorized into: five oral premalignant cases [leukoplakia] and 25 cases of invasive SCC with six cases presented by metastatic cervical lymph nodes. Paraffin embedded tissues were processed and sections were immunohistochemically stained using Avidin Biotin peroxidase complex [ABC] with Cox-2 and VEGF [Ab-7] epitopes. Positive staining for Cox-2 was detected in 3 out of 5[60%] of precancerous oral lesions, but it appeared in 9 out of 25 [36%] of SCC cases. Total positivity for Cox-2 reached 40% [12 out of the 30 samples]. As regards VEGF immuno staining, positive reaction was detected in one out of 5 cases [20%] of precancerous lesions and in 12 out of 25 cases [48%] of malignant lesions. Total positive reaction for VEGF was seen in 13 out of the 30 samples examined [43%]. Expression of VEGF was almost of the same ratio in premalignant and grade I tumors [25% and 26%] respectively, but it was increased in grade II [85%] and grade III tumors [66%]. Correlation between VEGF staining and grading of the SCC showed a significant difference [p=0.018]. An interesting finding in this study, is the expression of both Cox-2 and VEGF markers in normal fibroblasts, inflammatory cells, minor salivary glandular epithelium as well as some endothelial cells. Co-expression of both Cox-2 and VEGF staining was significantly correlated [p=0.004]. The results of this study demonstrated a definite role for Cyclo-oxygenase and VEGF as two pro-angiogenic factors that proved to be existing in both oral premalignant and SCC tumors .The dysplastic epithelium nearby a tumor area or in leukoplakia lesion expressed both markers with increased ratio for Cox-2 than VEGF factor. There was a direct relationship between VEGF immunopositivity and increased SCC grade. Also,co-expression of Cox-2 and VEGF was significantly correlated. From the present study it can be concluded that, both Cox-2 and VEGF factors are expressed in oral premalignant as well as SCC lesions, but Cox-2 may have a role in early stages of tumorigenesis, while VEGF showed a clear existence in higher grades of SCC

Role of modulation of vascular endothelial growth factor and tumor necrosis factor-alpha in gastric ulcer healing in diabetic rats

To assess the role of modulation of vascular endothelial growth factor and tumor necrosis factor-alpha in gastric ulcer healing in streptozotocin [STZ]- induced diabetic rats. forty male rats were made diabetic by intraperitoneal [i.p] STZ infection and ten rats were injected i.p. by a single dose of saline and served as a control for group II Six weeks following STZ or saline injection, gastric ulcers were induced by serosal application of acetic acid. Three days after acetic acid application, rats were divided into: group I[normal control], group II [STZ-injected rats], groups III. IV and V [STZ-injected rats treated with insulin, insulin and phosphodiesterase [PDE] inhibitor [pentoxifylline] [PTX] and insulin and Hydroxymethylglutaryl Coenzyme A [HMG-CoA] reductase inhibitor [simvastatin] respectively, for seven days following acetic acid application. At the end of the experimental period, plasma glucose was measured. Gastric ulcer area as well as gastric tumor necrosis factor- alpha [TNF-alpha], vascular endothelial growth factor [VEGF] and haemoglobin [Hb] concentrations were determined. STZ-injection induced diabetes, evidenced by significant higher mean value in plasma glucose concentration in group II compared to that of the control group [I] Significant delay in ulcer healing could be observed, in the form of significant increase in gastric ulcer area in group II compared to the control group I. STZ-injection resulted in significant increase in gastric TNF-alpha as well as a significant decrease in gastric VEGF concentrations together with a significant decrease in gastric angiogenic response evidenced by a significant decrease in gastric Hb concentration in group II compared to the control group I. The use of insulin, as well as combinations of insulin and PTX or simvastatin caused a significant decrease in plasma glucose concentration as well as a significant increase in gastric ulcer healing [evidenced by a significant decrease in ulcer area], gastric VEGF and gastric Hb concentration as well as significant decrease in gastric TNF-alpha compared to group II. A significant difference in gastric ulcer area and gastric TNF-alpha could be observed between rat that received combinations of insulin and PTX or simvastatin compared to rats that received insulin only. A significant difference in gastric VEGF and Hb was also found between the group that received combination of insulin and simvastatin compared to the group that received insulin only. Experimental DM impairs ulcer healing, depending upon the increased release of proinflammatory cytokines [e.g. TNF-alpha] and the attenuation of angiogenesis Insulin reversed this impairment of ulcer healing in diabetic rats, mainly due to the enhancement of angiogenesis and reduction in expression of TNF-alpha in the ulcer area. Phosphodiesterase [PDE] inhibitor [pentoxifylline], via suppressing TNF-alpha and hydroxymethylglutaryl coenzyme A [HMG-CoA] reductase inhibitor [simvastatin]. via suppressing TNF-alpha and increasing VEGF, are beneficial in enhancing gastric ulcer healing. These findings support the notion that impairment of healing of gastric ulcers in DM results from impairment of angiogenic response of the gastric mucosa to injury together with upregulotion of gastric TNF-alpha and suggest the feasibility of a novel treatment strategy for patients in whom impairment of ulcer healing complication of DM