- Email: [email protected]
Angiogenesis and endothelial progenitor cells (EPCS): A promissing alternative in cardiovascular regenerative medicine
e74
Abstracts / Atherosclerosis 241 (2015) e72ee148
By endothelial dysfunction, primarily understood imbalance between the production of vasodilation, angio, prothrombotic, proliferative factors. Experimental investigations have established that the vWF content of 20, 40 and 60 days of the study was significantly increased by 50.8, 64.5 and 75.2 % compared with the intact group. Most expressed the contents of this factor have a 60 day study, as its content, compared with 20 and 40 days of 16.2 and 6.5 %, respectively, significantly higher. Also, the results showed a marked increase homocysteine in the blood of the dynamics of development of the metabolic syndrome. Installations increased by 20, 40 and 60 days in experiment 2,8; 3 and 3,4 times in comparison with intact animals. The most marked increase in homocysteine mounted on day 60 of the experiment. During this period of study GC content compared to 40 during the day was increased by 9.6%. Contents of endothelin-1 is increased at all study time, especially more pronounced increase its installed on day 60 of the experiment. Thus, the results of the research show the development of endothelial dysfunction in the dynamics of development of the metabolic syndrome, the most pronounced changes of these parameters is set at 60 days of the experiment.
EAS-0410. EFFECTS OF SINGLE NUCLEOTID POLYMORPHISMS IN VEGFR2 ON SHEAR STRESS ACTIVATED ENDOTHELIAL CELLS K. Urschel 1, N. Schacher 1, *, A. Winterpacht 2, F. Pasutto 2, S. Achenbach 1, B. Dietel 1. 1 Cardiology and Angiology, University Hospital Erlangen, Erlangen, Germany; 2 Institute of Human Genetics, University of Erlangen-Nuremberg, Erlangen, Germany
Aim: Saffron is a dried stigma of Crocus sativus L. traditionally known as a spice agent and is also used for several medicinal purposes. Studies on hyperlipidaemic animal models suggest that crocin, the bioactive compound of saffron have anti-atherogenic properties. However, crocin's response at a cellular level remains unclear.Hence, this study aim to investigate the effects of saffron and crocin on monocyte-endothelial cell interaction in-vitro. Methods: The cytotoxicity effect of saffron and crocin were determined by MTT assay. HCAECs (Lonza, USA) were stimulated with LPS and treated with saffron and crocin (Sigma,USA) at different concentrations ranging from 0.05 - 25.00mg/ml and 0.001 - 1.600 mg/ml respectively. They were incubated for 16 hours. U937 cell line (ATCC, USA) was added and incubated for 1 hour. 0.25% rose bengal was added and PBS containing 10% FBS was used to remove the unbound cells. Ethanol:PBS 1:1 solution was used to stop the reaction. The absorbance was measured by spectrophotometer. Results: Saffron and crocin concentrations of up to 25.00mg/ml and 1.600mg/ml respectively gave >85% cell viability. Treatment with saffron showed monocyte-endothelial cell interaction was reduced significantly at concentrations of 6.3, 12.5 and 25.00mg/ml with percentage inhibition of 6.0±2.0 , 7.6±0.5 9.2±2.3%, p ¼0.03, 0.019 and 0.005 respectively. Similarly, crocin also suppressed it at concentrations of 0.04, 0.08,1.600 mg/ml with greater percentage inhibition of 11.6±3.5, 11.2±0.6 and 18.7±5.1%, p¼0.023, 0.004 and 0.007 respectively. Conclusion: Both compounds reduce monocyte-endothelial cell interaction. However, crocin alone exerts more inhibitory effect suggesting its effectiveness as anti-atherogenesis than its crude form, saffron.
* Corresponding author. Background: Endothelial activation due to local changes in shear-stress pattern is one of the initial steps in the development of atherosclerosis. Vascular endothelial growth factor receptor-2 (VEGFR2), which is activated by VEGF and fluid shear stress, is important for intracellular endothelial mechanotransduction. Recent studies identified single nucleotide polymorphisms (SNPs) in VEGFR2-gene associated with coronary artery disease (CAD), hypertension and myocardial infarction. Methods: Two SNPs within the VEGFR2-gene (rs2305948C>T and rs1870377T>A) were genotyped in 84 samples of human umbilical vein endothelial cells (HUVECs) using TaqMan assay. Genotyped HUVECs were seeded in bifurcating flow-through slides and exposed to non-uniform shear-stress for 18h, followed by 2h stimulation with TNF-a. Thereupon endothelial activation was analyzed measuring expression of VCAM-1 and E-selectin. Expression levels of VEGFR2 were determined by immunofluorescence (shear-stress conditions) and Western Blot (basal expression). Results: SNP-genotyping revealed a high inhomogeneity for the different genotypes in our population; e.g. only 7 specimens out of 84 showed genotype A/A in rs1870377 whereas no sample exhibited the genotype T/T in rs2305948. SNP rs2305948 showed no differences in VEGFR2-expression, both basal (p¼0,74) and under non-uniform shear-stress conditions (p¼0,21) comparing T/T to C/T-genotype. However, expression of VCAM-1 and Eselectin was decreased in C/T-genotype. In SNP rs1870377 A/A-genotype showed a lower expression of VEGFR2 in both experimental settings, whereas endothelial activation was clearly decreased in samples with A/T-genotype. Conclusion: We show that the investigated SNPs in VEGFR2 have a notable effect on shear-stress related endothelial activation, supporting the idea of VEGFR2-SNPs being approved risk factors for cardiovascular disease.
EAS-0452. THE EFFECT OF SAFFRON & ITS BIOACTIVE COMPOUND; CROCIN AGAINST MONOCYTE e ENDOTHELIAL CELL INTERACTION IN HUMAN CORONARY ARTERIAL ENDOTHELIAL CELLS M. Alicezah*, R. Ahmad, T. Rahman, G.R.A. Froemming, H. Nawawi. Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Selangor, Malaysia
* Corresponding author.
EAS-0500. ANGIOGENESIS AND ENDOTHELIAL PROGENITOR CELLS (EPCS): A PROMISSING ALTERNATIVE IN CARDIOVASCULAR REGENERATIVE MEDICINE R. Toro 1, *, M. Quezada 2, M. Monsalvo 3, B. Ruiz-Estevez 3, V. Aragon 3, C. Leal 3, I. Tinoco 3, A. Barba 4, E. Segura 5, A. Barba 6, A. Mangas 5. 1 Medicine, Medicine School (Cadiz University), El Puerto de Santa Maria, Spain; 2 Cardiology, Cruz Roja Hospital, Madrid, Spain; 3 Internal Medicine, Puerta Del Mar Hospital, Cadiz, Spain; 4 Medicine, Granada University, Granada, Spain; 5 Medicine, Medicine School (Cadiz University), Cadiz, Spain; 6 Medicine, Cadiz University, Cadiz, Spain
* Corresponding author. Introduction and objectives: EPCs has been considered as an useful tool in cardiovascular regenerative medicine due to their role in angiogenesis and vascular repair. EPCs seems to promote vascular remodeling by replacement of damage mature endhothelial cell and/or secreting certain angiogenic factors at the injured tissue. Such mechanisms needs to be characterized. Our aims is to identify proteins differentially expressed in the EPCs pf atherosclerosis patients and healthy controls in order to provide potential candidates to promote vascular regeneration. Methods: EPCs were isolated form healthy donors or patients undergoing carotid endarterectomy and cultured following state-of-the-art protocols. Cellular characterization of EPCs markers was carried out by flow cytometry after at days 4 and 7after isolation. Then EPCs were lysed, reduced and sequentially digested by LysC and Trypsin and the resulting peptides were labelled. Data analysis was performed with ProteomeDiscoveres 1.4. Results and discussion: Derived from analysis 1196 proteins were identified, including 52 proteins which were up-regulated and 38 down regulated in the EPCs of atherosclerosis patients versus controls. Differentially expressed proteins seem to be involved in several pathways regulating angiogenesis, cell mobility and cell-cell interactions. The function of these protein could be the cog in the machine for vascular repair.
Abstracts / Atherosclerosis 241 (2015) e72ee148
By endothelial dysfunction, primarily understood imbalance between the production of vasodilation, angio, prothrombotic, proliferative factors. Experimental investigations have established that the vWF content of 20, 40 and 60 days of the study was significantly increased by 50.8, 64.5 and 75.2 % compared with the intact group. Most expressed the contents of this factor have a 60 day study, as its content, compared with 20 and 40 days of 16.2 and 6.5 %, respectively, significantly higher. Also, the results showed a marked increase homocysteine in the blood of the dynamics of development of the metabolic syndrome. Installations increased by 20, 40 and 60 days in experiment 2,8; 3 and 3,4 times in comparison with intact animals. The most marked increase in homocysteine mounted on day 60 of the experiment. During this period of study GC content compared to 40 during the day was increased by 9.6%. Contents of endothelin-1 is increased at all study time, especially more pronounced increase its installed on day 60 of the experiment. Thus, the results of the research show the development of endothelial dysfunction in the dynamics of development of the metabolic syndrome, the most pronounced changes of these parameters is set at 60 days of the experiment.
EAS-0410. EFFECTS OF SINGLE NUCLEOTID POLYMORPHISMS IN VEGFR2 ON SHEAR STRESS ACTIVATED ENDOTHELIAL CELLS K. Urschel 1, N. Schacher 1, *, A. Winterpacht 2, F. Pasutto 2, S. Achenbach 1, B. Dietel 1. 1 Cardiology and Angiology, University Hospital Erlangen, Erlangen, Germany; 2 Institute of Human Genetics, University of Erlangen-Nuremberg, Erlangen, Germany
Aim: Saffron is a dried stigma of Crocus sativus L. traditionally known as a spice agent and is also used for several medicinal purposes. Studies on hyperlipidaemic animal models suggest that crocin, the bioactive compound of saffron have anti-atherogenic properties. However, crocin's response at a cellular level remains unclear.Hence, this study aim to investigate the effects of saffron and crocin on monocyte-endothelial cell interaction in-vitro. Methods: The cytotoxicity effect of saffron and crocin were determined by MTT assay. HCAECs (Lonza, USA) were stimulated with LPS and treated with saffron and crocin (Sigma,USA) at different concentrations ranging from 0.05 - 25.00mg/ml and 0.001 - 1.600 mg/ml respectively. They were incubated for 16 hours. U937 cell line (ATCC, USA) was added and incubated for 1 hour. 0.25% rose bengal was added and PBS containing 10% FBS was used to remove the unbound cells. Ethanol:PBS 1:1 solution was used to stop the reaction. The absorbance was measured by spectrophotometer. Results: Saffron and crocin concentrations of up to 25.00mg/ml and 1.600mg/ml respectively gave >85% cell viability. Treatment with saffron showed monocyte-endothelial cell interaction was reduced significantly at concentrations of 6.3, 12.5 and 25.00mg/ml with percentage inhibition of 6.0±2.0 , 7.6±0.5 9.2±2.3%, p ¼0.03, 0.019 and 0.005 respectively. Similarly, crocin also suppressed it at concentrations of 0.04, 0.08,1.600 mg/ml with greater percentage inhibition of 11.6±3.5, 11.2±0.6 and 18.7±5.1%, p¼0.023, 0.004 and 0.007 respectively. Conclusion: Both compounds reduce monocyte-endothelial cell interaction. However, crocin alone exerts more inhibitory effect suggesting its effectiveness as anti-atherogenesis than its crude form, saffron.
* Corresponding author. Background: Endothelial activation due to local changes in shear-stress pattern is one of the initial steps in the development of atherosclerosis. Vascular endothelial growth factor receptor-2 (VEGFR2), which is activated by VEGF and fluid shear stress, is important for intracellular endothelial mechanotransduction. Recent studies identified single nucleotide polymorphisms (SNPs) in VEGFR2-gene associated with coronary artery disease (CAD), hypertension and myocardial infarction. Methods: Two SNPs within the VEGFR2-gene (rs2305948C>T and rs1870377T>A) were genotyped in 84 samples of human umbilical vein endothelial cells (HUVECs) using TaqMan assay. Genotyped HUVECs were seeded in bifurcating flow-through slides and exposed to non-uniform shear-stress for 18h, followed by 2h stimulation with TNF-a. Thereupon endothelial activation was analyzed measuring expression of VCAM-1 and E-selectin. Expression levels of VEGFR2 were determined by immunofluorescence (shear-stress conditions) and Western Blot (basal expression). Results: SNP-genotyping revealed a high inhomogeneity for the different genotypes in our population; e.g. only 7 specimens out of 84 showed genotype A/A in rs1870377 whereas no sample exhibited the genotype T/T in rs2305948. SNP rs2305948 showed no differences in VEGFR2-expression, both basal (p¼0,74) and under non-uniform shear-stress conditions (p¼0,21) comparing T/T to C/T-genotype. However, expression of VCAM-1 and Eselectin was decreased in C/T-genotype. In SNP rs1870377 A/A-genotype showed a lower expression of VEGFR2 in both experimental settings, whereas endothelial activation was clearly decreased in samples with A/T-genotype. Conclusion: We show that the investigated SNPs in VEGFR2 have a notable effect on shear-stress related endothelial activation, supporting the idea of VEGFR2-SNPs being approved risk factors for cardiovascular disease.
EAS-0452. THE EFFECT OF SAFFRON & ITS BIOACTIVE COMPOUND; CROCIN AGAINST MONOCYTE e ENDOTHELIAL CELL INTERACTION IN HUMAN CORONARY ARTERIAL ENDOTHELIAL CELLS M. Alicezah*, R. Ahmad, T. Rahman, G.R.A. Froemming, H. Nawawi. Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Selangor, Malaysia
* Corresponding author.
EAS-0500. ANGIOGENESIS AND ENDOTHELIAL PROGENITOR CELLS (EPCS): A PROMISSING ALTERNATIVE IN CARDIOVASCULAR REGENERATIVE MEDICINE R. Toro 1, *, M. Quezada 2, M. Monsalvo 3, B. Ruiz-Estevez 3, V. Aragon 3, C. Leal 3, I. Tinoco 3, A. Barba 4, E. Segura 5, A. Barba 6, A. Mangas 5. 1 Medicine, Medicine School (Cadiz University), El Puerto de Santa Maria, Spain; 2 Cardiology, Cruz Roja Hospital, Madrid, Spain; 3 Internal Medicine, Puerta Del Mar Hospital, Cadiz, Spain; 4 Medicine, Granada University, Granada, Spain; 5 Medicine, Medicine School (Cadiz University), Cadiz, Spain; 6 Medicine, Cadiz University, Cadiz, Spain
* Corresponding author. Introduction and objectives: EPCs has been considered as an useful tool in cardiovascular regenerative medicine due to their role in angiogenesis and vascular repair. EPCs seems to promote vascular remodeling by replacement of damage mature endhothelial cell and/or secreting certain angiogenic factors at the injured tissue. Such mechanisms needs to be characterized. Our aims is to identify proteins differentially expressed in the EPCs pf atherosclerosis patients and healthy controls in order to provide potential candidates to promote vascular regeneration. Methods: EPCs were isolated form healthy donors or patients undergoing carotid endarterectomy and cultured following state-of-the-art protocols. Cellular characterization of EPCs markers was carried out by flow cytometry after at days 4 and 7after isolation. Then EPCs were lysed, reduced and sequentially digested by LysC and Trypsin and the resulting peptides were labelled. Data analysis was performed with ProteomeDiscoveres 1.4. Results and discussion: Derived from analysis 1196 proteins were identified, including 52 proteins which were up-regulated and 38 down regulated in the EPCs of atherosclerosis patients versus controls. Differentially expressed proteins seem to be involved in several pathways regulating angiogenesis, cell mobility and cell-cell interactions. The function of these protein could be the cog in the machine for vascular repair.