Rabbit Vein Research Articles

Pretreatment of isolated vein with rapamycin nanoparticles inhibits vein graft stenosis in rabbits

To explore the effects of pretreatment of carbopol-encapsulated rapamycin-loaded nanoparticles (RPM-NP) on vein graft stenosis in a rabbit vein graft model. A segment of common carotid artery was replaced with a segment of external jugular vein in 40 rabbits. They were separated into four treatment groups, i.e. Group A: vein grafts were pretreated with intraluminal RPM-NP perfusion; Group B: peripheral venous veins were injected with RPM-NP; Group C: vein grafts received an equivalent perfusion of empty vehicle; Group D: vein grafts received no treatment. At Day 28 post-operation, the grafts and normal veins were harvested for histological examinations to analyze the indicators of intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index. At Day 28 post-operation, the intimal/media thickness ratios were 0.26 ± 0.02, 0.73 ± 0.05, 0.71 ± 0.04, 0.69 ± 0.03 and 0.24 ± 0.01 in Groups A, B, C and D and the normal vein; the collagen volume index 0.24 ± 0.03, 0.56 ± 0.06, 0.53 ± 0.07, 0.49 ± 0.08 and 0.21 ± 0.01 respectively. Compared with the normal veins, the pathological indicators of vein graft intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index had significant differences in Groups B, C and D (all P < 0.05). But there were no significant differences among 3 groups (all P > 0.05). Compared with the normal vein, the parameters of vein graft intimal thickness, internal diameter, intimal/media thickness ratio and collagen volume index had no significant difference in Group A (all P > 0.05). But as compared with other groups, these indicators had statistical significant difference in Group A (all P < 0.05). The local pretreatment of isolated vein with rapamycin nanoparticles may inhibit neointimal hyperplasia and prevent effectively vein graft stenosis.

Distribution and Permeability of Capillaries at the Skin of the Conception Vessel and the Governor Vessel in Healthy Rabbits

To investigate the distribution and permeability of blood vessels on the Conception Vessel and the Governor Vessel in the physiological state. Evans blue (EB) solution was injected into the marginal ear vein of healthy rabbits. Three hours after injection, the rabbits were sacrificed and the skin on the Conception Vessel and the Governor Vessel and the corresponding bilateral non-channels was collected. EB was extracted with 7:3 acetone: physiological saline, and the absorbance of EB at each skin tissue was measured with a spectrophotometer. The A value of EB absorbance at the Conception Vessel on the abdominal skin was lower than that of the corresponding bilateral non-channels with a statistically significant difference (P< 0.01). The A value of EB absorbance at the Governor Vessel on the back was higher than that of the corresponding bilateral non-channels (P < 0.05). There was no statistical difference in the A value of EB absorbance between the bilateral non-channels of the abdomen and the back (P > 0.05). There were differences in capillary distribution and permeability between the Conception Vessel, the Governor Vessel and the corresponding bilateral non-meridians.

Histamine and H1-histamine receptors faster venous circulation

The study has analysed the action of histamine in the rabbit venous system and evaluated its potential role in contraction during increased venous pressure. We have found that a great variety exists in histamine sensitivity and H1-histamine receptor expression in various types of rabbit veins. Veins of the extremities (saphenous vein, femoral vein, axillary vein) and abdomen (common iliac vein, inferior vena cava) responded to histamine by a prominent, concentration-dependent force generation, whereas great thoracic veins (subclavian vein, superior vena cavas, intrathoracic part of inferior vena cava) and a pelvic vein (external iliac vein) exhibited slight sensitivity to exogenous histamine. The lack of reactivity to histamine was not due to increased activity of nitric oxide synthase (NOS) or heme oxygenase-1. H1-histamine receptor expression of veins correlated well with the histamine-induced contractions. Voltage-dependent calcium channels mediated mainly the histamine-induced force generation of saphenous vein, whereas it did not act in the inferior vena cava. In contrast, the receptor-operated channels were not involved in this response in either vein. Tyrosine phosphorylation occurred markedly in response to histamine in the saphenous vein, but not in the inferior vena cava. Histamine induced a prominent ρ kinase activation in both vessels. Protein kinase C and mitogen-activated protein kinase (MAPK) were not implicated in the histamine-induced intracellular calcium sensitization. Importantly, transient clamping of the femoral vein in animals caused a short-term constriction, which was inhibited by H1-histamine receptor antagonist in vivo. Furthermore, a significantly greater histamine immunopositivity was detected in veins after stretching compared to the resting state. We conclude that histamine receptor density adapts to the actual requirements of the circulation, and histamine liberated by the venous wall during increased venous pressure contributes to the contraction of vessels, providing a force for the venous return.

Mineralocorticoid receptor expression in human venous smooth muscle cells: a potential role for aldosterone signaling in vein graft arterialization.

Experimental studies have suggested a role for the local renin-angiotensin-aldosterone system in the response to vascular injury. Clinical data support that aldosterone, via activation of the mineralocorticoid receptor (MR), is an important mediator of vascular damage in humans with cardiovascular disease. In mineralocorticoid-sensitive target tissue, aldosterone specificity for MR is conferred enzymatically by the cortisol-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11βHSD2). However, the role of MR/aldosterone signaling in the venous system has not been explored. We hypothesized that MR expression and signaling in venous smooth muscle cells contributes to the arterialization of venous conduits and the injury response in vein bypass grafts. MR immunostaining was observed in all samples of excised human peripheral vein graft lesions and in explanted experimental rabbit carotid interposition vein grafts, with minimal staining in control greater saphenous vein. We also found upregulated transcriptional expression of both MR and 11βHSD2 in human vein graft and rabbit vein graft, whereas control greater saphenous vein expressed minimal MR and no detectable 11βHSD2. The expression of MR and 11βHSD2 was confirmed in cultured human saphenous venous smooth muscle cells (hSVSMCs). Using an adenovirus containing a MR response element-driven reporter gene, we demonstrate that MR in hSVSMCs is capable of mediating aldosterone-induced gene activation. The functional significance for MR signaling in hSVSMCs is supported by the aldosterone-induced increase of angiotensin II type-1 receptor gene expression that was inhibited by the MR antagonist spironolactone. The upregulation of MR and 11βHSD2 suggests that aldosterone-mediated tissue injury plays a role in vein graft arterialization.

Acute Outflow Obstruction of Hepatic Veins in Rabbits

To compare time-enhancement curve parameters of contrast-enhanced sonography in acute partial hepatic venous outflow obstruction with those of a baseline study. Contrast-enhanced sonography was performed in 11 rabbits with bolus administration of a sulfur hexafluoride contrast agent (0.1 mL/kg). After baseline scanning for 3 minutes, a 5.3F balloon catheter was placed into the left hepatic vein. Obstruction was artificially induced by 0.4-mL balloon inflation, and sonography was repeatedly performed thereafter. On images stored with 1-second intervals, 2 × 2-mm regions of interest were placed, and mean luminosity was measured. Time-enhancement curves were plotted, and contrast arrival times, peak enhancement values, peak enhancement times, 50% wash-out times, and 3-minute wash-out rates were obtained. Paired t tests were performed to evaluate the significance of differences in the parameters between baseline and obstruction. On baseline sonography, the median contrast arrival time, peak enhancement value, peak enhancement time, 50% wash-out time, and 3-minute wash-out rate were 6 (range, 4-8; mean ± SD, 5.9 ± 1.2) seconds, 188.5 (104.7-209.5; 178.4 ± 33.1) arbitrary units, 19 (14-27; 19.8 ± 4.1) seconds, 75 (60-101; 78.2 ± 13.9) seconds, and 89.7% (81.3%-95.1%; 88.4% ± 4.9%), respectively. With obstruction, those values were 7 (5-12; 6.9 ± 2.3) seconds, 202.8 (98.2-215.1; 186.0 ± 39.3) arbitrary units, 31 (17-59; 32 ± 11.6) seconds, 101 (47-136; 96.2 ± 23.6) seconds, and 79.2% (66.2%-88.8%; 79.1% ± 7.6%). Compared with baseline, the peak enhancement time was significantly delayed from 19 to 31 seconds (P = .0027), 50% wash-out time significantly delayed from 75 to 101 seconds (P = .0209), and 3-minute wash-out rate significantly decreased from 89.7% to 79.2% (P < .0001) with obstruction, but there were no significant differences in contrast arrival times and peak enhancement values (P = .0756 and .2179). Contrast-enhanced sonography can provide quantitative assessment of microbubble congestion in partial hepatic venous outflow obstruction. The peak enhancement time and 50% wash-out time are delayed and 3-minute wash-out rate is decreased in rabbits with artificially induced obstruction compared with a baseline study.

Intravascular glucose/lactate sensors prepared with nitric oxide releasing poly(lactide- co-glycolide)-based coatings for enhanced biocompatibility

Intravenous amperometric needle-type enzymatic glucose/lactate sensors intended for continuous monitoring are prepared with a novel nitric oxide (NO) releasing layer to improve device hemocompatibility. To create an underlying NO release coating, the sensors with immobilized enzymes (either glucose oxidase or lactate oxidase) are prepared with a thin layer of poly(lactide- co-glycolide) (PLGA) loaded with lipophilic diazeniumdiolate species that slowly release NO via a proton driven reaction. An outer thin layer (ca. 30 μm) of PurSil (polyurethane/dimethylsiloxane copolymer) limits the flux of glucose and lactate to the inner layer of enzyme, to provide the desired linear amperometric response. A 30 μm coating of PLGA containing 33 wt% of the appropriate NO donor ( N-diazeniumdiolated dibutylhexanediamine, DBHD/N 2O 2) can release NO at a physiologically relevant rate >1 × 10 −10 mol min −1 cm −2 for at least 7 days without influencing the analytical performance of the glucose/lactate sensors. In vitro, the sensors exhibit relatively stable amperometric response over a one-week period with high selectivity over interferences (e.g., ascorbic acid) required for blood monitoring applications. Glucose sensors implanted in the veins of rabbits for 8 h exhibit significantly enhanced hemocompatibility for the NO release sensors vs. corresponding controls (without NO release in same animals), with greatly reduced thrombus formation on their surfaces. Further, the analytical performance of the NO release glucose sensors are superior to controls placed in the veins of the same animals, with a greater accuracy in measuring blood glucose levels as evaluated using a Clarke error grid type analysis.

Pulmonary Enhancement Imaging with Dual Energy CT for the Detection of Pulmonary Embolism in a Rabbit Model: Comparison to Perfusion Planar Scintigraphy, SPECT and SPECT-CT Modalities

Pulmonary enhancement imaging (PEI) derived from dual-energy computed tomographic (CT) imaging has been used to detect perfusion defects from pulmonary embolism (PE). The purpose of this study was to compare the ability of PEI, planar, single photon-emission CT (SPECT) perfusion scintigraphy, and SPECT-CT fusion images to detect perfusion defect in a PE rabbit model. A PE model was made by injecting Gelfoam into the femoral veins of rabbits (n = 16). After 2 hours, 16 experimental rabbits and three control rabbits underwent contrast-enhanced dual-energy CT scans, from which PEI and CT pulmonary angiography were created, and planar, SPECT, and SPECT-CT fusion images were then obtained and evaluated. Pathologic determination of locations and numbers of lung lobes with PE were recorded. The sensitivity and specificity of the above-mentioned modalities were calculated using the histopathologic results as reference standards. Emboli were present in 31 pulmonary lobes and absent in 64 lung lobes in histopathologic analysis. With the histopathologic findings as the gold standard, sensitivities and specificities of PEI, planar, SPECT, and SPECT-CT fusion images to detect PE were 100% and 96.9%, 71.0% and 84.4%, 77.4% and 90.6%, and 74.2% and 93.8%, respectively. McNemar's tests showed that PEI had higher diagnostic accuracy for the detection of PE than three scintigraphic images (all P values < .05), while three scintigraphic images had similar diagnostic accuracy (all P values = NS). This study demonstrates that PEI from dual-energy CT imaging can provide higher diagnostic accuracy for detecting PE than planar, SPECT, and SPECT-CT fusion images in a rabbit model.

Tissue-type plasminogen activator gene targets thrombolysis in atriums

Our previous investigations showed that retroviral gene transfer of tissue-type plasminogen activator (tPA) effectively targeted thrombolysis in vitro and in the model of inferior caval veins of rabbits. This study is to identify the target thrombolysis of retroviral vector recombinant pLEGFP-N1-tPA transferred into the tissue around the Dacron patch (the same materials making of the ring of mechanical valve) in left atriums of rabbits. 70 Dacron patches were transplanted into the left atriums of 70 New Zealand white rabbits. The rabbits were randomly divided into three groups according to the different handling methods, including local pLEGFP-N1-tPA transferred group (gene therapy group, 30 animals), pLEGFP-N1 transferred group (control group, 20 animals), medium DMEM+10% neonate calf serum (NCS) injected group (blank control group, 20 animals). Samples of blood, Dacron pieces and left atriums (auricles) wall from half of above in each group were harvested on second day and another half were harvested on 75th day after surgery. The EGFP expression of harvested left atriums (auricles) wall were observed under the confocal. The thrombi on the surface of Dacron patches were detected by stereoscope and electron microscope. The tPA expression in left atriums (auricles) wall and in blood from left atriums were detected by Western blot and their thrombolysis and activities were observed and calculated in plasma plates. ELISA were used to identify the contents of tPA. No thrombus was seen on the surface of Dacron patches that were transplanted in left atriums by tPA locally transferring around them. Activity and content of tPA were high in local tissue of left atrium and in blood of left atrium. It demonstrated effectively thrombolysis by tPA rapidly, efficiently and long expressing. This puts the foundation of mechanical valve replacement model for tPA gene valve, next.

The effect of Na+‐Ca2+ exchange on the recovery from Ca2+ dependent inactivation of L‐type Ca2+ channel in cardiomyocytes in pulmonary vein

Ca2+ dependent inactivation (CDI) of L‐type Ca2+ channel (CaL) is well known phenomena and Ca2+ release from SR greatly affected CaL kinetics, which was known as the release dependent inactivation (RDI). We would like to see whether L‐type Ca2+ kinetics were changed by Na+‐Ca2+ exchange (NCX) which would control the subsarcolemmal Ca2+. We used the isolated cardiomyocytes in main pulmonary vein of rabbit. RDI was induced by applying 2.5 msec prepulse from ‐40 mV to 10 mV and then the same amplitude of 50 msec test pulse was applied with various intervals. In the presence of 0.1 mM EGTA in the pipette solution, the inactivation tau (itau) was increased and the peak current amplitude (PCA) was decreased. As the interval was increased, itau and PCA were progressively returned to control ICaL. In the absence of bath Na+, the initial change was much larger and the recovery process was prolonged. In the presence of BAPTA 10 mM in the pipette solution, these phenomena were disappeared. Ryanodine or thapsigargin also attenuate these phenomena. In conclusion, NCX is an important regulator for the recovery of CaL from CDI by controlling subsarcolemmal Ca2+. (supported by NRF, 2009‐0076234)

Prolonged hypercholesterolemia-induced tissue factor expression in rabbit vein grafts: a potential mechanism for graft failure

To evaluate tissue factor (TF) expression in vein grafts interposed in the arterial circulation of hypercholesterolemic rabbits. Veins implanted in the arterial circulation of normocholesterolemic rabbits respond by inflammation and infiltration by monocytes with transient TF expression. In a hypercholesterolemic milieu these monocytes may differentiate into macrophages capable of enhanced TF synthesis, which may facilitate hyperplasia and thrombosis. Autologous jugular veins interposed in the carotid artery of hypercholesterolemic rabbits were harvested at 1, 2, 4, 6, and 8 weeks after surgery and examined for presence and localization of rabbit TF antigen. Protein extracted from vein segments was evaluated for procoagulant activity by bioassay and for TF protein content by western blotting. Rabbit TF antigen was observed mostly in the subendothelium of vein grafts. Peak TF procoagulant activity observed at 1-2 weeks postsurgery (2.3+/-1.8 pg/mg, P<0.006) declined to 0.9+/-0.5, 0.2+/-0.1, and 0.15+/-0.06 pg/mg at 4, 6, and 8 weeks, respectively (P<0.03). Western blotting showed a time-dependent pattern for rabbit TF protein with prolonged expression peaking at 6 weeks. Prolonged expression of biologically active rabbit TF and TF protein were shown within jugular vein grafts of hypercholesterolemic rabbits. This response, reported for the first time and attributed to increased cholesterol levels, may possibly contribute to enhanced hyperplasia. These results suggest that TF expression could serve as another mechanism underlying vein graft failure and that hypercholesterolemia in bypass patients should be treated aggressively beginning within the weeks after surgery.

C-Jun Regulates Shear- and Injury-inducible Egr-1 Expression, Vein Graft Stenosis after Autologous End-to-Side Transplantation in Rabbits, and Intimal Hyperplasia in Human Saphenous Veins

Coronary artery bypass graft failure represents an unsolved problem in interventional cardiology and heart surgery. Late occlusion of autologous saphenous vein bypass grafts is a consequence of neointima formation underpinned by smooth muscle cell (SMC) migration and proliferation. Poor long term patency and the lack of pharmacologic agents that prevent graft failure necessitate effective alternative therapies. Our objective here was to evaluate the effect of targeted inhibition of the bZIP transcription factor c-Jun on intimal hyperplasia in human saphenous veins and vein graft stenosis after autologous end-to-side transplantation. DNAzymes targeting c-Jun attenuated intimal hyperplasia in human saphenous vein explants. Adenovirus-forced c-Jun expression stimulated SMC proliferation, proliferating cell nuclear antigen, and MMP-2 expression. c-Jun DNAzymes abrogated Adeno-c-Jun-inducible SMC growth and wound repair and reduced intimal thickening in jugular veins of New Zealand white rabbits 4 weeks after autologous end-to-side transplantation to carotid arteries. Conversely, in a DNAzyme-free setting, Adeno-c-Jun potentiated neointima formation in the veins compared with Adeno-LacZ. Inducible c-Jun expression is ERK1/2- and JNK-dependent but p38-independent. Injury- and shear-inducible c-Jun controls early growth response-1. These data demonstrate that strategies targeting c-Jun may be useful for the prevention of vein graft stenosis. Control of one important shear-responsive transcription factor by another indicates the existence of transcriptional amplification mechanisms that magnify the vascular response to cell injury or stress through inducible transcriptional networks.

Pretreatment with intraluminal rapamycin nanoparticle perfusion inhibits neointimal hyperplasia in a rabbit vein graft model

PurposePoly lactic-co-glycolic acid nanoparticles (PLGA-NP) are widely used as a biodegradable biomaterial in medicine. Rapamycin-eluting stents have been used for prevention of restenosis during surgery. This study investigated the effect of pretreatment with intraluminal perfusion of carbopol-encapsulated rapamycin-loaded PLGA nanoparticles (RAP-PLGA-NP) on neointimal hyperplasia in a rabbit vein graft model.MethodsA segment of common carotid artery was replaced with a segment of external jugular vein in 60 rabbits which were then separated into four treatment groups, ie, Group 1, in which vein grafts were pretreated with intraluminal RAP-PLGA-NP perfusion, Group 2 in which vein grafts underwent equivalent empty vehicle (PLGA-NP) perfusion, Group 3, in which vein grafts received no treatment, and Group 4, which served as a sham operation group receiving normal vein contrast. On postoperative day 28, the grafts and normal veins were harvested for histologic examination, flow cytometry analysis, and high-performance liquid chromatography measurement.ResultsCompared with Group 1, the intima of the grafts were thickened, the ratio of intimal area to vessel area increased, and the collagen volume index of the vein grafts increased significantly in Groups 2 and 3. The cell proliferation index in Group 1 (21.11 ± 3.15%) was much lower than that in Group 2 (30.35 ± 2.69%) and in Group 3 (33.86 ± 8.72%). By high-performance liquid chromatography measurement, retention of rapamycin was detected in Group 1 (11.2 ± 0.37 μg/10 mg) 28 days after single drug perfusion.ConclusionPretreatment with intraluminal RAP-PLGA-NP perfusion may inhibit neointimal hyperplasia in vein grafts by penetrating into local tissue and limiting cell proliferation.

Chronic treatment of hydroxytryptamine type 2a receptor antagonist sarpogrelate hydrochloride modulates the vasoreactivity of serotonin in experimental rabbit vein grafts

It has been suggested that 5-hydroxytryptamine (5-HT) plays a role in the pathogenesis of vein graft spasms. It is suggested that smooth muscle 5-HT(2A) and 5-HT(1B) receptors contribute to 5-HT-induced contraction, while endothelial 5-HT(1B) receptors contribute to the 5-HT-induced endothelium-mediated relaxation. We recently found that chronic administration of the selective 5-HT(2A) receptor antagonist sarpogrelate hydrochloride (SH) enhances the function of endothelium-derived nitric oxide (NO) in rabbit vein grafts. However, it is unknown if such treatment modulates 5-HT-induced vasospasm in vein grafts, and if so, what the underlying mechanisms are. Male rabbits were divided into two groups: a control group and an SH-treated group. The jugular vein was interposed in the carotid artery in reversed fashion. Isometric tension was examined using vein grafts after 4 weeks. 5-HT (10(-8) -10(-6) M)-induced contraction was obtained in each group in the absence or presence of the NO synthase inhibitor l-N(G)-nitroarginine (L-NNA). The expression of 5-HT(2A) and 5-HT(1B) receptors was examined immunohistochemically. The 5-HT induced a concentration-dependent contractions in both groups. L-NNA did not significantly modify the 5-HT-induced contraction in the control group but enhanced it in the SH group. The 5-HT(1B) receptor antagonist GR55562 inhibited the 5-HT-induced contraction in the control group, while it increased the sensitivity of contraction to 5-HT in the SH-treated group in the absence (but not in the presence) of L-NNA. Positive immunoreactivities against 5-HT(1B) and 5-HT(2A) receptors were identified in endothelial and medial regions of vein grafts in both groups, and the expression of 5-HT(2A) receptors (but not 5-HT(1B) receptors) was significantly less in the SH-treated group than in the control group. Chronically administered SH to rabbits upregulates the autoinhibitory mechanism by 5-HT through a release of NO from endothelium via an activation of endothelial 5-HT(1B) receptors, thus attenuating its own contraction in vein grafts. Furthermore, such SH treatment downregulates the expression of smooth muscle 5-HT(2A) receptors, thus further attenuating the 5-HT-induced contraction. These novel findings further support the clinical usefulness of SH in vein graft spasm after bypass grafting.

Detection of pulmonary embolism by dual energy CT: correlation with perfusion scintigraphy and histopathological findings in rabbits

The purpose of the study was to compare the ability of dual energy CT (DECT) and perfusion scintigraphy (PS) to detect pulmonary embolism (PE) in a rabbit model. Gelfoam (n = 20) or saline (n = 4) was injected into the femoral vein of rabbits. After 2 h, DECT pulmonary angiography (CTPA) was used to create blood flow imaging (BFI) and fusion images. The rabbits then underwent PS. Pathological determination of locations and numbers of lung lobes with PE was recorded. The sensitivity and specificity for BFI, CTPA, fused images and PS were calculated using the pathological results as reference standards. Compared with pathological evaluation, CTPA correctly identified PE in 40 lobes and absence of emboli in 80 lobes, corresponding to a sensitivity and specificity of 100%. BFI and fused images correctly identified PE in 40 lobes and the absence of emboli in 78 lobes, corresponding to a sensitivity and specificity of 100% and 98%, respectively. PS correctly detected 27 lobes with PE and 65 lobes without PE, corresponding to a sensitivity and specificity of 68% and 81%, respectively. BFI, CTPA and fused images derived from a single contrast-enhanced DECT provide a higher diagnostic accuracy of detecting PE than PS in a rabbit model.

Sarpogrelate hydrochloride reduced intimal hyperplasia in experimental rabbit vein graft

The selective 5-HT(2A) receptor antagonist sarpogrelate has been clinically used for treatment in atherosclerotic diseases. However, it remains unknown whether administration of sarpogrelate inhibits intimal hyperplasia seen in autologous vein grafts. Therefore, we sought to clarify this question using an experimental rabbit vein graft model. Male rabbits were divided into two groups: a control group and a sarpogrelate-treated group. The jugular vein was interposed in the carotid artery in reversed fashion for 4 weeks and intimal hyperplasia of the grafted vein was measured (n = 8, in each group). Acetylcholine (ACh)-induced endothelium-dependent relaxation was tested by precontraction with prostaglandin F(2alpha) (PGF(2alpha), 5 muM) (n = 5, in each). endothelial nitric oxide synthase (eNOS) protein expression and superoxide production of these veins were also assessed. The suppression of intimal hyperplasia was significantly greater in the sarpogrelate-treated group than in the control group. ACh induced an endothelium-dependent relaxation in the sarpogrelate-treated group (but not in the control group). In endothelium-intact strips from the sarpogrelate-treated group, the nitric oxide (NO) synthase inhibitor nitroarginine enhanced the PGF(2alpha)-induced contraction and blocked the ACh-induced relaxation. Immunoreactive eNOS protein expression was similar between the two groups but superoxide production (estimated from ethidium fluorescence) in endothelial cells was significantly smaller in the sarpogrelate-treated group. The present results indicate that in vivo blockade of 5-HT(2A) receptors leads to an inhibition of intimal hyperplasia in rabbit vein graft. It is suggested that an increased function of endothelium-derived NO through a reduction in endothelial superoxide production may be a possible underlying mechanism for this. These novel findings support the clinical usefulness of sarpogrelate for preventing intimal hyperplasia in vein graft after bypass grafting.

Recombinant Adeno-Associated Virus-Based Gene Transfer of Cathelicidin Induces Therapeutic Neovascularization Preferentially via Potent Collateral Growth

Therapeutic neovascularization is a concept well validated in animal models, however, without clear-cut success in clinical studies. To achieve prolonged transgene expression, recombinant adeno-associated virus (rAAV) was used in a chronic ischemic hind-limb model and the human antimicrobial peptide cathelicidin (LL-37/hCAP-18) was used as proangiogenic factor. Seven days after femoral artery excision, 0.5 x 10(11) rAAV particles encoding for green fluorescent protein (rAAV.GFP), cathelicidin (rAAV.cath), or vascular endothelial growth factor A (rAAV.VEGF-A) were retroinfused into the anterior tibial vein of rabbits (n = 5 per group). In addition, one rAAV.cath-treated group obtained a constant infusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin into the ischemic tissue starting on day 7. On day 7 and day 35 angiography of both hind limbs was performed for collateral quantification and frame count score (cinedensitometry). Capillary-to-muscle fiber ratios were obtained on day 35. Compared with controls, application of rAAV.cath induced a gain of perfusion (153 +/- 12 vs. 107 + 9% of day 7 controls) via increased collateral growth (length index, 161 +/- 14 vs. 97 +/- 9%, controls), but no significant capillary growth (1.16 +/- 0.09 vs. 0.99 +/- 0.08, controls). Wortmannin application completely abolished the effects of rAAV.cath, indicating the involvement of the PI3K signal pathway. In conclusion, rAAV-mediated cathelicidin expression is capable of inducing functionally relevant neovascularization, preferentially by collateral growth. The rAAV-based vectors as long-expressing vector expression systems and cathelicidin as proangiogenic factor provide a promising new combination in the treatment of peripheral artery disease.

Effect of corticosteroids on phlebitis induced by intravenous infusion of antineoplastic agents in rabbits

Phlebitis caused by intravenous infusion of antineoplastic agents is one of the critical problems when anticancer therapy is prolonged. We have already reported that both rapid infusion and dilution of the injection solution were effective methods for reducing phlebitis caused by vinorelbine (VNR) in rabbits. The aim of this study was to explore other practical methods for preventing phlebitis caused by VNR and doxorubicin (DXR) in a rabbit model. VNR is often used with cisplatin, and dexamethasone (DEX) has been co-administered for prevention of cisplatin-induced nausea. DXR is used with prednisolone (PSL) in the CHOP regimen for the treatment of non-Hodgkin's lymphoma. Therefore, the present study investigated the prevention of phlebitis due to VNR with DEX and that due to DXR with PSL. VNR and DXR were diluted with normal saline to prepare test solutions at concentrations of 0.6 mg/mL and 1.4 mg/mL, respectively. Each test solution was infused into the auricular veins of rabbits. Two days after VNR infusion and three days after DXR infusion, the veins were evaluated histopathologically. The effect of DEX on VNR-induced phlebitis was evaluated by infusion of DEX before or after VNR. The effect of PSL on DXR-induced phlebitis was similarly evaluated by co-infusion of PSL. The histopathological features of phlebitis caused by the antineoplastic agents differed between VNR and DXR: VNR did not cause the loss of venous endothelial cells, but caused inflammatory cell infiltration, edema, and epidermal degeneration. In contrast, DXR caused the loss of venous endothelial cells and chrondrocyte necrosis. Pre-treatment and post-treatment with DEX significantly decreased VNR-induced phlebitis compared with the control group and pre-treatment was particularly effective. Co-infusion of PSL also significantly decreased phlebitis caused by DXR, but its effect was less marked. The present findings suggested that pre-treatment with DEX may be a useful method for preventing phlebitis due to VNR, and that co-infusion of PSL has the potential to prevent phlebitis caused by DXR.

Abstract 247: Therapeutic Neovascularization by AAV2/9 Based Gene Transfer of hVEGF-A and hPDGF in Chronic Ischemia (rabbit model)

Therapeutic neovascularization by several growth factors is a concept well established in multiple animal models, albeit lacking clearcut clinical success to date. Therefore, we modified our therapeutic approach by combining the potent angiogenic agent hVEGF-A with hPDGF, a growth factor known for attracting pericytes and smooth muscle cells for arterial vessel maturation. In order to enhance efficacy, we utilized adeno-associated virus (AAV) 2/9, allowing for prolonged transgene expression. A lower hVEGF-AAV-concentration was used to avoid hemangiomaformation. 7 days after femoral artery excision, 1×10e11 hVEGF-A-AAV alone or in combination with 1×10e12 hPDGF-AAV particles were retroinfused into the anterior tibial vein of rabbits (n=5/group, p&lt;0.05). LacZ-AAV2 was used as reporter-gene controls. At d7 and 35 angiography of both hindlimbs was performed for collateral quantification (% of d7 level) and frame count score (cinedensitometry, % of d7 level). Capillary density (Capillary muscle fiber ratio, CM/F) was assessed at d35 by PECAM-1 and alkaline-phosphatase-staining of the ischemic gastrocnemic and tibial anterior muscles, pericyte-quantification was assessed by NG2-staining. LacZ-expression was detected at d35 in control animals. hVEGF-A-AAV retroinfusion strongly induced angiogenesis (CM/F 1,32+0,07 vs. 0.96+0,08, controls), but failed to enhance collateral growth (125+7 vs. 95+6%, controls, p&lt;0.05) or perfusion (frame count score: 136+12% vs. 107+9%, p=0.07). hVEGF-A/hPDGF co-application, however, enhanced perfusion (163+8%, p&lt;0.05) via an increased collateral growth (146+9%, p&lt;0.01) at a similar capillary density (1,44+0,10). We conclude, that efficacy of hVEGF-A-transfection is increased by hPDGF-co-transfection with a long-acting AAV. Utilizing this approach, the main target of hPDGF appears to be the collateral vasculature, complementary to the capillary growth induced by hVEGF-A. Thus, the combination of hVEGF-A and hPDGF combined with a long-acting AAV-vector is a promising tool in the quest of efficient therapeutic neovascularization.

Intravascular site-specific delivery of a therapeutic antisense for the inhibition of restenosis

Background Polymeric nanoparticles (NPs) have been implicated as potential gene carriers in the treatment of various genetic and acquired diseases. In this work we investigated the efficacy of NPs as gene carrier for intravascular gene therapy in animal models of restenosis. Methods A therapeutic antisense against the monocyte chemotactic protein-1 (anti-MCP-1) was encapsulated into the poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) using double-emulsion/solvent evaporation technology. Laser defractometer was used to assess size distribution of NPs. Particle morphology was assessed by scanning electron microscopy (SEM). DNA content in NPs was determined by DNA extraction using TE buffer from a chloroform solution dissolved a known amount of NPs. DNA concentration was assayed by spectrophotometer. In vitro DNA release was performed in the TE buffer at 37 °C utilizing double-chamber diffusion cell. NPs loaded with pEGFP and anti-MCP-1 gene were tested in SMC cell culture for transduction efficiency. The anti-MCP-1 NPs were further evaluated in rabbit vein grafting and carotid artery injury models for their potential in inhibition of restenosis. Results The NPs demonstrated a steady in vitro release of DNA with approximately 95% of total enclosed DNA released within 30 days. Anti-sense MCP-1 expression was confirmed in arterial tissues with single infusion of the therapeutic NPs into the injured rabbit carotid arteries. The intima/media ratio of arteries treated with the anti-MCP-1 NP was reduced by 43% compared with control groups following a 2-week treatment. In a rabbit jugular vein-to-artery-bypass grafting model, animals with local-infusion of anti-MCP-1 NPs also demonstrated a significantly lower intimal hyperplasia than that of control groups with no or free antisense treatment. Conclusion Collectively, our data revealed that local delivered anti-MCP-1 NPs effectively inhibited experimental restenosis.

Ablation of High Intensity Focused Ultrasound Combined with SonoVue on Rabbit VX2 Liver Tumors: Assessment with Conventional Gray-Scale US, Conventional Color/Power Doppler US, Contrast-Enhanced Color Doppler US, and Contrast-Enhanced Pulse-Inversion Harmonic US

We investigated effects of high intensity focused ultrasound (HIFU) combined with contrast agent SonoVue on rabbit VX2 liver tumors by using conventional gray-scale ultrasonography (US), color/power Doppler (CD/PD) US, contrast-enhanced color Doppler (CE CD) US, and contrast-enhanced pulse-inversion harmonic (CE PIH) US. Fourteen days after implantation of VX2 tumors in livers of 50 rabbits, animals were randomly separated into two groups. Based on principles of HIFU, the volume of the tumor was divided into several parallel "planes" to be ablated. Before ablation on each "plane," 0.2 mL SonoVue was injected in bolus via ear veins of rabbits in group II and normal saline solution was administrated in group I. Conventional gray-scale US, CD US, PD US, CE CD US, and CE PIH US were performed before and after ablation. Twenty-three surviving rabbits in each group underwent HIFU ablation. Conventional gray-scale US showed ablated areas diffusely hyperechoic. On CE PIH US, coagulated areas presented perfusion defect. Both conventional gray-scale US and CE PIH US showed the ablated volume in group II was larger than that in group I. CD US and PD US demonstrated residual vessels in periphery ablated areas in group I, but no residual vessels in group II. CE CD US and CE PIH US depicted less residual vessels in periphery ablated areas in group II than that in group I. By enlarging ablated volume of tissue and reducing residual vessels, effects of HIFU ablation on rabbit VX2 liver tumors were enhanced by contrast agent SonoVue.