Characterization of the cytokine profile of platelet rich plasma (PRP) and PRP-induced cell proliferation and migration: Upregulation of matrix metalloproteinase-1 and -9 in HaCaT cells / 대한혈액학회지

BACKGROUND: The underlying rationale of platelet rich plasma (PRP) therapy is that an injection of concentrated PRP at the site of injury may promote tissue repair via cytokine release from platelets. The molecular mechanisms of PRP therapy in the skin wound healing process are not well understood at present, and would benefit from clarification. METHODS: PRP was stimulated with angonists for 5 min, and cytokine profile analysis was performed. To investigate the wound healing activity of PRP, cell proliferation and migration analyses were performed in skin cells. The effects of PRP were analyzed on the expression and activity of matrix metalloproteinase (MMP)-1, -2, -9, and the activation of transcription factors. RESULTS: Thrombin was found to be a strong stimulator of PRP activation to release growth factors and chemokines. PRP induced cell proliferation and migration in HUVECs, HaCaT cells, and HDFs, as well as MMP-1and MMP-9 expression in HaCaT cells, but PRP did not have a significant effect on the expression or activity of MMPs in HDFs. The transcription factors, including signal transducer and activator of transcription-3 (STAT-3) were found to be phosphorylated following PRP treatment in HaCaT cells. CONCLUSION: In this study, we have identified the cytokine profile of activated PRP after agonist stimulation. We have shown that PRP plays an active role in promoting the proliferation and migration of skin cells via the regulation of MMPs, and this may be applicable to the future development of PRP therapeutics to enhance skin wound healing.

Biological effects of fucoidan isolated from Fucus vesiculosus on thrombosis and vascular cells / 대한혈액학회지

BACKGROUND: Fucoidan is a highly sulfated glycosaminoglycan, which has a molecular structure similar to that of heparin. The antithrombotic effects of fucoidan in vitro have been widely reported, but its antithrombotic effects in vivo as well as its other biological properties in vitro have not been well investigated. METHODS: This study investigated the effects and mechanism of fucoidan from Fucus vesiculosus on thrombosis both in vitro and in vivo. A ferric chloride-induced mouse carotid artery thrombosis model was used to determine the antithrombotic effects of fucoidan in vivo. Additionally, changes in the levels of proinflammatory cytokines and chemokines were examined in vascular cells treated with fucoidan. RESULTS: In vivo studies employing a ferric chloride-induced mouse carotid artery thrombosis model indicated that fucoidan had a stronger antithrombotic activity than heparin. Further, vascular cells treated with fucoidan demonstrated a decrease in proinflammatory cytokine and chemokine production as well as inhibition of proliferation. CONCLUSION: The major findings of this study showed that fucoidan has a stronger antithrombotic effect than heparin in vivo and that fucoidan has an inhibitory effect on proinflammatory cytokine production and proliferation of vascular cells.

Targeted Ultrasound Imaging of Apoptosis with Annexin A5 Microbubbles in Acute Doxorubicin-Induced Cardiotoxicity

BACKGROUND: The aim of this study was to assess the feasibility of targeted ultrasound imaging on apoptosis with annexin A5 microbubbles (A5MB) in acute doxorubicin-induced cardiotoxicity. METHODS: Avidinated and octafluoropropan-filled phospholipid microbubbles were conjugated with biotinylated annexin A5. To confirm the specific binding of A5MB, flow cytometry was performed with hydrogen peroxide induced apoptosis in rat aorta smooth muscle cells incubated with fluorescein-5-isothiocyanate (FITC) labeled annexin A5 and A5MB. Adult male rats were injected intraperitoneally with 5 mg/kg doxorubicin weekly for 3 weeks (n = 5). Control rats were injected with normal saline (n = 5). At 24 hours after the final treatment, triggering imaging was performed 15 min after an intravenous bolus injection of A5MB for washout of freely circulating microbubbles. After echocardiography, the heart was isolated for histological detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. RESULTS: In the in vitro tests, fluorescence intensity was low for healthy cells and high for apoptotic cells when incubated with FITC-labeled annexin A5 and A5MB. Rats treated with doxorubicin showed significant contrast opacification of the myocardium on contrast echocardiography using A5MB. However, no opacification was observed in control rats. Apoptosis was confirmed by TUNEL assay in doxorubicin treated rats. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with targeted ultrasound imaging using A5MB in rats.

Up-regulation of Interleukin-8 by Vascular Endothelial Growth Factor in Vasculatures in vivo / 대한혈액학회지

BACKGROUND: Vascular endothelial growth factor (VEGF) plays an essential role in promoting angiogenesis during tumor development. In addition, VEGF can mediate the inflammatory response in tumors. VEGF increases the level of neutrophil migration by upregulating interleukin-8 (IL-8) in endothelial cells in vitro. However, it is unclear if VEGF can mediate IL-8 production in vivo. METHODS: To address this issue, this study examined the effect of VEGF on IL-8 production in vivo using an adenovirus transduction and mouse ear assay. RESULTS: Adenovirus-encoded VEGF (VEGF-Ad) increased the level of IL-8 production in endothelial cells in vitro compared to the control-adenovirus (CTL-Ad). The mouse ear assay showed that VEGF-Ad increased the level of IL-8 production in the endothelium. Immunohistochemistry showed that the IL-8 proteins were expressed in the vasculature within a human glioblastoma, which is known to strongly express VEGF. CONCLUSION: These results suggest that VEGF can mediate the inflammatory response in endothelial cells in vivo via the up-regulation of IL-8.

The Effects of the beta2-Adrenergic Receptor Gene Polymorphism on the Risk of Essential Hypertension

BACKGROUND AND OBJECTIVES: Considering the physiological importance of the beta2-adrenergic receptor (ADRB2) gene, "functional" molecular variations of the gene might cause attenuated vasodilatation, leading to increased total peripheral resistance and; hence, ultimately result in hypertension. Significant evidence has been provided for the pathophysiological involvement of the beta2-adrenergic receptor (ADRB2) in hypertension. The genetic variation of the ADRB2 gene, to see if there might be any relationship to essential hypertension, was investigated. SUBJECTS AND METHODS: One ADRB2 gene polymorphism, Arg16Gly (Arg->Gly variant), was investigated in this study. The genotypes of Arg16Gly in 318 hypertensive patients and 309 normotensive subjects were analyzed. RESULTS: No significant differences were found in the allele and genotype frequencies between patients with hypertension and normotensive subjects. There was no association of the ADRB2 polymorphism (Arg16Gly) with hypertension or the other phenotypes measured in our study populations.CONCLUSION: Our data suggest that ADRB2 Arg16Gly polymorphisms are unlikely to confer the genetic susceptibility for hypertension in the Korean population. However, further investigation is warranted to clarify the relevance of ADRB2 polymorphisms in blood pressure regulation.

Transdifferentiation of Cultured Bovine Lens Epithelial Cells into Myofibroblast-like Cells by Serum Modulation

An after-cataract is caused by the proliferation of residual cells over the equator of the lens. These cells subsequently migrate to the posterior lens capsule, where they undergo aberrant differentiation into fiber-like cells or transdifferentiation into fibroblast-like cells. To study the precise molecular mechanisms of transdifferentiation, an attempt was made to establish an in vitro system, in which the lens epithelial cells (LECs) of the pre-equatorial zone could be transdifferentiated into fibroblast-like cells. The required conditions for culturing the LECs were identified as consisting of four phases; intact bovine explants, explant-cultured, serum-modulated and additionally modulated LECs. The LECs of each phase were compared by examining changes in the expression of the epithelial-mesenchymal transition (EMT) -related genes and changes in cellular morphology and adhesion. The explants that were cultured in a medium containing 10% fetal bovine serum (FBS) for 2 weeks, showed changes in the expression of the EMT-related genes, although the other explant-cultured cells maintained an epithelial morphology. To introduce a transition into mesenchymal cells, the explant cultures were subcultured in a medium containing 20% FBS for six passages. These cells displayed an elongated morphology and were able to grow and migrate in a similar way to fibroblast cells. The expression of the EMT-related genes, such as, extracellular matrix proteins and integrins, was altered. This was similar to the alteration of the 3-dimensional collagen gels model previously reported. During a further process of EMT by additional serum modulation, the inhibitory effect of disintegrin on cell adhesion was gradually decreased, integrin expression was differentially regulated and alpha-smooth muscle actin was post-translationally modified from the point of passage number six. Overall, it can be concluded that terminal transdifferentiation accompanies changes in the cytoskeletal proteins and cell surface molecules. These are modulated in systematic patterns of post-transcriptional and post-translational regulation and patterns of gene regulation, by the synergic effects of several transforming factors contained in serum. Therefore, posterior capsular opacification may also be accompanied by this molecular mechanism.

Development of New Coronary Stent-Grafts using Surface-Modified Polymers

BACKGROUND AND OBJECTIVE: The purpose of this study was to develop a new type of coronary stent-graft, using surface modification of polymeric synthetic graft materials, to improve biocompatibility. MATERIALS AND METHODS: Three different polymers, Dacron, GoreTex and Teflon were tested. During the surface-modification process, hydroxybutyl acrylate (HBA) choline, an excellent blood-compatible phospholipid, was stably grafted onto the polymer surface. The optimal conditions for maximizing the amount of HBA choline grafted onto the polymeric surface were determined by quantitative analysis. The surface-modified polymers were then tested for their biocompatibility using an in vitro platelet adhesion test. Thereafter, stent-grafts were constructed with each of three different types of surface-modified polymer and implanted in porcine coronary arteries to compare their biocompatibility in vivo. RESULTS: In the platelet adhesion test, all the surface-modified polymers showed better biocompatibility than the control polymers. The in vitro biocompatibility correlated positively with the increasing quantity of grafted HBA choline. In the animal experiment, the surface-modified Teflon stent-graft showed the best biocompatibility. Whereas, all pigs implanted with the modified Dacron and GoreTex stent-grafts died within 48 hours of the implantation, five out of the six pigs with the Teflon stent-grafts remained alive at after the 4th week. In four of the five surviving pigs, angiography, intravascular ultrasound (IVUS) and histological evaluations demonstrated the patency of the stent-grafts, with a uniform neointima formation covering the entire stent-graft, without stent thrombosis or chronic inflammatory cells. CONCLUSION: The surface-modified Teflon coronary stent-grafts showed good in vitro and in vivo biocompatibility. Further animal and clinical studies will be required to validate the efficacy of the surface-modified polymer stent-grafts.

Effect of Curcuminoids and Natural Plants Extract Mixture on the Cardiovascular System in Rats

Antiplatelet aggregation, anticoagulant and lipid-lowering drugs are clinically widely used for secondary preventive purpose in the cardiovascular patients, but there is no primary preventive agents to prevent these diseases. With the aim of developing effective primary agents for cardiovascular diseases, we tried to formulate an optimized mixture of natural plants extract containing Theae sinensis, Camelliae sinensis, Vitis vinifera, Gingko folium and curcuminoids from Curcuma longa and to evaluate its anti-thrombotic and anti-hypercholesterolemic effects in vivo. The inhibitory effect of curcuminoids on vascular smooth muscle cell proliferation and migration were also investigated in vitro. in the animal experiments treated with hyperlipidemic diet, oral treatment of curcuminoids and natural plants extracts mixture (100 mg/kg) into male Sprague Dawley rats for 7 week simultaneously inhibited platelet aggregation as well as improved lipid profile in the blood. Compared to control group, both of curcuminoids-treated and mixture-treated groups revealed significantly decrease of total cholesterol (24.4%, 28.6%), free cholesterol (25.1%, 24.0%), cholesterol ester (14.6%, 29.0%), LDL-cholesterol (27.0%, 32.0%) and triglyceride (15.0%, 31.0%), respectively. However, both groups showed increase of HDL-cholesterol (46.6% and 51.5%) . In particular, atherogenic index of curcuminoids and mixture treatment group was significantly decreased to 47.0% and 56.0%, respectively. Furthermore, oral treatment of curcuminoids and mixture significantly inhibited collagen-induced platelet aggregation (21.1% and 29.1%, respectively), compared to control group. The anti-thrombotic values of mixture was almost similar to that of aspirin treatment (100 mg/kg) group. These results suggest that the oral treatment of curcuminoids-based natural plant extract mixture improved cardiovascular conditions in hyperlipidemic rats.

Enhancement of Thrombolytic Therapy by Transcutaneous Ultrasound and Perfluorocarbon Exposed Sonicated Dextrose Albumin in Thrombotic Arterial Occlusion

BACKGROUND AND OBJECTIVES: Perfluorocarbon exposed sonicated dextrose albumin (PESDA) microbubbles has been suggested to facilitate thrombus disruption under the transcutaneous ultrasound (US). Thus, we investigated whether such a noninvasive approach could augment thrombolytic effect of fibrinolytic agent in an experimental thrombotic model. MATERIALS AND METHODS: Thrombus formation was induced with electrical injury in the rabbit iliofemoral arteries (n=20): Thrombus occlusion was documented by angiography in all arteries. In the control group, only tissue plasminogen activator (t-PA, 3 mg/kg) was administered intrav-enously in five rabbits. In the Group 1 (n=9), injured arteries were exposed to transcutaneous US (20 kHz, 30 W/cm2, continuous mode) with t-PA (3 mg/kg). In the Group 2 (n=6), the same treatment was given while administering PESDA continuously (10 ml/min, intravenous). Angiographic results were evaluated at 10 minute interval for 1 hour respectively. RESULTS: In the control group, two of five iliofemoral arteries (40.0%) were recanalized and one of nine iliofemoral arteries (11.1%) was recanalized in Group 1. In contrast, four of six iliofemoral arteries (66.7%) were recanalized angiographically in Group 2 (p=0.392 vs. control group: p=0.047 vs. Group 1). However, late reocclusion occurred in all iliofemoral arteries of Group 2. CONCLUSION: Although PESDA with transcutaneous US significantly enhanced initial angiographic patency rate of t-PA, it was associated with high rate of reocclusion. Further studies will be necessary for clinical application of this noninvasive method in acute arterial occlusion.

Antiplatelet Effect of Hirudin in a Rabbit Carotid Artery Eversion Model

BACKGROUND AND OBJECTIVES: Thrombin and its interaction with platelets play a pivotal role in arterial thrombus formation. Hirudin, an anticoagulant agent derived from medicinal leeches(Hirudo medicinalis), is a unique and specific thrombin inhibitor with no effect on other serine protease. We investigated the inhibitory effect of hirudin on platelet deposition in a rabbit carotid artery eversion model of acute arterial thrombosis. MATERIALS AND METHODS: The everted arterial segments were perfused with 111 Indium-labeled human platelets only(control, n=8), and a mixed solution of 111 Indium-labeled human platelets and hirudin(30, 45, 60, 90 microgram/ml, n=3, respectively). Platelet deposition was calculated by a gamma-counter and confirmed by scanning electron microscopy. RESULTS: 1) Indium-111 labeling efficiency of platelets was 87.0+/-6.6%, and the aggregation of platelets was not changed after labeling. The number of platelets perfused through each arterial segment was 4.3 +/-0.2x10(8) platelets/ml. 2) The control group showed a platelet deposition rate of 23.9+/-7.0 % and a number of platelet deposition of 9.8+/-2.5x10(8) platelets/cm2 . 3) Platelet deposition of arteries perfused with hirudin(60 microgram/ml) was significantly decreased compared with that of the control group(2.9+/-0.6 vs 9.8+/-2.5x10(8)/cm2 , p<0.05). 4) The number of deposited platelets in hirudin-perfused arteries was dose-dependently decreased(30 microgram/ml:6.7+/-1.4x10(8) /cm2 , 45 microgram/ml: 4.8+/-1.7x10(8) /cm2 , 60 microgram/ml: 2.9+/-1.8x10(8)/cm2, 90 microgram/ml:2.9+/-1.4x10(8)/cm2: p<0.05 vs. control, respectively). 5) Scanning electron microscopic examination revealed significantly reduced platelet deposition in hirudin-perfused groups compared with control group. CONCLUSION: Hirudin inhibits effectively platelet deposition and arterial thrombus formation in a rabbit carotid artery eversion model. The antiplatelet effect of hirudin in this model suggests that hirudin may be an useful antithrombotic agent therapeutically useful in the prevention of acute arterial thrombus formation.

The Effect of Estrogen Containing Liposome Local Delivery on the Neointimal Hyperplasia in the Rat Carotid Artery Balloon-Injury Model

BACKGROUND AND OBJECTIVES: Estrogen has been reported to inhibit migration and proliferation of vascular smooth muscle cells in vitro and in vivo. Sustained local delivery represents a potential alternative to systemic administrationbecauseitcan achieve higher tissue drug levels at site of balloon injury avoiding systemic side effects. We investigated the effect and mechanism of nanoparticulate sustained-release carrier system using liposome incorporating 17beta-estradiol (E2) on neointimal formation in rat carotid artery balloon injury model. MATERIALS AND METHODS: 17-estradiol benzoate, egg phosphatidylcholine, cholesterol, polyethyleneglycol-phosphatidylethanolamine were mixed to produce E2 -liposome formula where the final concentrations of lipids and E2 were 10 mg/ml and 66 M, respectively. The size of the particle was less than 200 nm. Rat carotid artery balloon injury model was used with Sprague-Dawley rats weighing 350+/-30g. Rats were divided into 3 groups of saline (n=22), liposome (n=46) and E2-liposome (n=46) and received 0.2 ml of each agent at injured site. 1) Rats from all groups were sacrificed at 7 (n=4), 14 (n=6), and 21 (n=12) days after injury, respectively. Morphometric analysis was performed for calculating medial area, neointimal area and I/M (intimal area/medialarea)ratio2)Rats from liposome and E2-liposome group sreceived 100mg/kg of 5-bromo-2'-deoxyuridine (BrdU) at 25, 9 and 1hr before sacrifice at 1 (n=4), 3 (n=4), 7 (n=4), and 14 (n=4) days after injury. BrdU and proliferating cell nuclear antigen (PCNA) stains were performed to elucidate a mechanism of inhibitory effect of E2. RESULTS: 1) There was no increase in the neointimal area in liposome group compared with saline group at 7, 14, and 21 days after injury, respectively. 2) There was 17%, 30%, and 34% reduction of I/M ratio in E2 -liposome group compared with liposome group at 7, 14 and 21 days after injury, respectively. 3) BrdU and PCNA stain revealed that at day 3, labelling index (LI) of media was lower in E2-liposome than in liposome group (p<0.05), and at day 7, LI of neointima was not significantly different between the two groups despite smaller neointimal area in the E2-liposomegroup. CONCLUSION: Nanoparticulateliposomeformula appears to be biocompatible. Local intraluminal infusion of E2 liposome formula after balloon injury of rat carotid artery significantly decreased neointimal formation. The mechanism seems to be the inhibitory effect on the proliferative response of smooth muscle cells in media at an early stage of injury. This formula appears to show potential for clinical applications in the prevention of neointimal formation following balloon angioplsty.

The Inhibitory Effect of Triflusal (Disgren)on the Platelet Aggregation in Healthy Volunteers: Impedance Method with the Whole Blood

BACKGROUND: Antiplatelet drugs play an important role in the prevention and treatment of coronary artery diseases. Triflusal, an antiplatelet drug structually related to acetylsalicylic acid, selectively inhibits the cyclooxygenase of platelet and thromboxane A2 formation. However there is a controversy about the clinical dosage and the quantitative evaluation of the platelet antiaggregatory effect of triflusal. In this study we have evaluated the platelet antiaggregatory effect and cost-effective dosage of triflusal in the whole blood of healthy volunteers. METHODS: Using the whole blood of 50 healthy people, we performed a baseline platelet aggregation function test induced by adenosine diphosphate (ADP) and collagen. The subjects were subdivided into 3 treated groups (300 mg, 600 mg, 900 mg). We compared the platelet aggregation effect between the baseline results and 2 weeks after triflusal administration. RESULTS: Triflusal inhibited the platelet aggregation induced by ADP and collagen in the 600 mg administration group most effectively. The platelet aggregation induced by collagen was inhibited dose-dependently. The definite inhibitory responders (% inhibition > or = 25) for platelet aggregation induced by collagen were more common than those induced by ADP (33% vs 27% in 300 mg, 71% vs 53% in 600 mg, 78% vs 39% in 900 mg). There were no serious clinical side-effects except gastrointestinal trouble. One volunteer in the 900 mg treated group discontinued the treatment due to epigastric pain. CONCLUSION: We conclude that triflusal has a dose-dependent inhibitory effect on platelet aggregation induced by collagen and that the most effective dosage for platelet antiaggregation effect is 600 mg per day.

The Inhibitory Effect of Triflusal (Disgren)on the Platelet Aggregation in Healthy Volunteers: Impedance Method with the Whole Blood

BACKGROUND: Antiplatelet drugs play an important role in the prevention and treatment of coronary artery diseases. Triflusal, an antiplatelet drug structually related to acetylsalicylic acid, selectively inhibits the cyclooxygenase of platelet and thromboxane A2 formation. However there is a controversy about the clinical dosage and the quantitative evaluation of the platelet antiaggregatory effect of triflusal. In this study we have evaluated the platelet antiaggregatory effect and cost-effective dosage of triflusal in the whole blood of healthy volunteers. METHODS: Using the whole blood of 50 healthy people, we performed a baseline platelet aggregation function test induced by adenosine diphosphate (ADP) and collagen. The subjects were subdivided into 3 treated groups (300 mg, 600 mg, 900 mg). We compared the platelet aggregation effect between the baseline results and 2 weeks after triflusal administration. RESULTS: Triflusal inhibited the platelet aggregation induced by ADP and collagen in the 600 mg administration group most effectively. The platelet aggregation induced by collagen was inhibited dose-dependently. The definite inhibitory responders (% inhibition > or = 25) for platelet aggregation induced by collagen were more common than those induced by ADP (33% vs 27% in 300 mg, 71% vs 53% in 600 mg, 78% vs 39% in 900 mg). There were no serious clinical side-effects except gastrointestinal trouble. One volunteer in the 900 mg treated group discontinued the treatment due to epigastric pain. CONCLUSION: We conclude that triflusal has a dose-dependent inhibitory effect on platelet aggregation induced by collagen and that the most effective dosage for platelet antiaggregation effect is 600 mg per day.

Report of a case: A case of medically resistant diabetic gangrene of foot which is healed by HELP (Heparin-mediated extracorporeal low density-lipoprotein cholesterol fibrinogen precipitation) therapy / 대한내과학회지

HELP (Heparin-mediated extracorporeal low-density lipoprotein fibrinogen precipitation) is an LDL apheresis system which has been utilized as a last therapeutic option for drug resistant hypercholesterolemia. Recently the scope of the treatment has been expanded to coronary artery obstructive disease (CAOD) or peripheral arterial obstructive disease (PAOD) with dyslipidemia. We applied six sessions of HELP therapy with a week interval to a patient with diabetic gangrene of both feet and PAOD who had elevated LDL-cholesterol and fibrinogen despite maximal drug therapy. Lipids, Lp (a) and fibrinogen were measured on plasma samples before and after treatment. Changes of symptoms and physical findings were reported before, immediately after and 3 month after treatment. In every session, LDL cholesterol level was reduced more than 40%. Mean LDL cholesterols were reduced from 133.5 mg/dL to 55.0 mg/dL (59%). Total cholesterol (104.5 mg/dL;51% decrease), triglycerides (142.0 mg/dL;47% decrease) and Lp (a) (24.3 mg/dL; 58% decrease) levels were also reduced. Mean HDL cholesterol was reduced to 6.3 mg/dL (25%) with prompt recovery to pretreatment level within one week. Mean fibrinogen decreased from 571.0 mg/dL to 253.3 mg/dL (58%) without bleeding complications. Two episodes of dizziness with spontaneous resolution were observed during or over three days after treatment. Two sessions of HELP made diabetic gangrene on toes improved. After six sessions, the gangrenes showed near-complete healing. So we report a case of a patient who had persistent diabetic gangrene of both feet despite proper revascularization, which was completely healed by HELP without significant side effects.