Novel microspheres reduce the formation of deep venous thrombosis and repair the vascular wall in a rat model.

: L-Arginine (L-arg), widely known as a substrate for endogenous nitric oxide synthesis, can improve endothelial function associated with the vasculature, inhibit platelet aggregation, and alter the activity of vascular smooth muscle cells. P-selectin is a membrane component of the platelet alpha-granule and the endothelial cell-specific Wiebel-Palade body that plays a central role in mediating interactions between platelets and both leukocytes and the endothelium. The experiment was designed to evaluate the effect of novel microspheres with L-arg targeting P-selectin on the formation of deep vein thrombosis and repair of vascular wall in a rat model. Thrombosis of the inferior vena cava was induced by applying a piece of filter paper (5 mm × 10 mm) saturated with 10% FeCl3 solution for 5 min. Targeted microspheres with L-arg, targeted microspheres with water, and saline were injected into the tail veins of the rats after 30 min of applying the filter paper saturated with 10% FeCl3 solution. The dry weight and length of the thrombus isolated from the inferior vena cava were significantly decreased in the group with L-arg in microsphere after 24 h. No significant differences in prothrombin time, activated partial thromboplastin time, thrombin time, and fibrinogen among the groups were indicated. Images revealed that apoptosis in the vascular wall was less in the group injected with targeted microspheres with L-arg than in the other two groups at 1 and 8 d postsurgery. Meanwhile, cell proliferation was considerably excessive in the group injected with L-arg wrapped in targeted microspheres. Therefore, these novel microspheres could decrease the formation of thrombus in the early stages and in the subsequent periods of thrombosis. The microspheres can also enhance the vitality of impaired endothelial cells and reduce cell apoptosis.

Divergent signaling mechanisms for venous versus arterial contraction as revealed by endothelin-1.

OBJECTIVE: Venous function is underappreciated in its role in blood pressure determination, a physiologic parameter normally ascribed to changes in arterial function. Significant evidence points to the hormone endothelin-1 (ET-1) as being important to venous contributions to blood pressure. We hypothesized that the artery and vein should similarly depend on the signaling pathways stimulated by ET-1, specifically phospholipase C (PLC) activation. This produces two functional arms of signaling: diacylglycerol (DAG; protein kinase C [PKC] activation) and inositol trisphosphate (IP3) production (intracellular calcium release). METHODS: The model was the male Sprague-Dawley rat. Isolated tissue baths were used to measure isometric contraction. Western blot and immunocytochemical analyses measured the magnitude of expression and site of expression, respectively, of IP3 receptors in smooth muscle/tissue. Pharmacologic methods were used to modify PLC activity and signaling elements downstream of PLC (IP3 receptors, PKC). RESULTS: ET-1-induced contraction was PLC dependent in both tissues as the PLC inhibitor U-73122 significantly reduced contraction in aorta (86% ± 4% of control; P < .05) and vena cava (49% ± 11% of control; P < .05). However, ET-1-induced contraction was not significantly inhibited by the IP3 receptor inhibitor 2-aminoethoxydiphenylborane (100 µM) in vena cava (82% ± 8% of control; P = .23) but was in the aorta (55% ± 4% of control; P < .05). All three IP3 receptor isoforms were located in venous smooth muscle. IP3 receptors were functional in both tissues as the novel membrane-permeable IP3 analogue (Bt-IP3; 10 µM) contracted aorta and vena cava. Similarly, whereas the PKC inhibitor chelerythrine (10 µM) attenuated ET-1-induced contraction in vena cava and aorta (5% ± 2% and 50% ± 5% of control, respectively; P < .05), only the vena cava contracted to the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. CONCLUSIONS: These findings suggest that ET-1 activates PLC in aorta and vena cava, but vena cava contraction to ET-1 may be largely IP3 independent. Rather, DAG­not IP3­may contribute to contraction to ET-1 in vena cava, in part by activation of PKC. These studies outline a fundamental difference between venous and arterial smooth muscle and further reinforce a heterogeneity of vascular smooth muscle function that could be taken advantage of for therapeutic development.

Reverse-mode Na+/Ca2+ exchange is an important mediator of venous contraction.

The Na(+)/Ca(2+) exchanger (NCX) is a bi-directional regulator of cytosolic Ca(2+), causing Ca(2+) efflux in forward-mode and Ca(2+) influx in reverse-mode. We hypothesized that reverse-mode NCX is a means of Ca(2+) entry in rat aorta (RA) and vena cava (RVC). NCX protein in RA and RVC was confirmed by immunoprecipitation. To assess NCX function, isometric contraction and intracellular Ca(2+) was measured in RA and RVC rings in response to low extracellular Na(+), endothelin-1 (ET-1), and KCl, in the presence or absence of the NCX antagonist KB-R7943. In RVC, low extracellular Na(+) caused vasoconstriction and an increase in intracellular Ca(2+) that was attenuated by 10µM KB-R7943. KB-R7943 (10 µM) attenuated maximal contraction to ET-1 in RVC (53 ± 9% of control), but not RA (91±1% of control). KB-R7943 (10 µM) reduced the maximal contraction to KCl in RA (48 ± 5%) and nearly abolished it in RVC (9 ± 2%), suggesting that voltage-dependent Ca(2+) influx may be inhibited by KB-R7943 as well. However, the L-type Ca(2+) channel inhibitor nifedipine (1 µM) did not alter ET-1-induced contraction. Our findings suggest that reverse-mode NCX is an important mechanism of Ca(2+) influx in RVC but not RA, especially during ET-1-induced contraction. Also, the effects of KB-R7943 on ET-1-induced contraction of RA and RVC are predominantly mediated by reverse-mode NCX inhibition and not due to off-target inhibition of Ca(2+) channels.

C1-esterase inhibitor protects against early vein graft remodeling under arterial blood pressure.

OBJECTIVES: Arterial pressure induced vein graft injury can result in endothelial loss, accelerated atherosclerosis and vein graft failure. Inflammation, including complement activation, is assumed to play a pivotal role herein. Here, we analyzed the effects of C1-esterase inhibitor (C1inh) on early vein graft remodeling. METHODS: Human saphenous vein graft segments (n=8) were perfused in vitro with autologous blood either supplemented or not with purified human C1inh at arterial pressure for 6h. The vein segments and perfusion blood were analyzed for cell damage and complement activation. In addition, the effect of purified C1inh on vein graft remodeling was analyzed in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery. RESULTS: Application of C1inh in the in vitro perfusion model resulted in significantly higher blood levels and significantly more depositions of C1inh in the vein wall. This coincided with a significant reduction in endothelial loss and deposition of C3d and C4d in the vein wall, especially in the circular layer, compared to vein segments perfused without supplemented C1inh. Administration of purified C1inh significantly inhibited vein graft intimal thickening in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery. CONCLUSION: C1inh significantly protects against early vein graft remodeling, including loss of endothelium and intimal thickening. These data suggest that it may be worth considering its use in patients undergoing coronary artery bypass grafting.

[Comparative characteristic of energy supply disturbances in arterial and venous walls of rabbits with alloxan diabetes and monoiodacetate intoxication].

We showed here that energy metabolism in thoracic rabbit aorta and posterior vena cava is disturbed two weeks following the induction of alloxan-induced diabetes and monoiodacetate intoxication. In the vessels studied, the alterations are manifested in the decrease in the intensity of glucose uptake, oxygen consumption, lactic acid production, and ATP resynthesis. Simultaneously, the ATP content is significantly reduced. The possible significance of these disorders in the development of Monckeberg's arteriosclerosis is discussed.

Topical HDL administration reduces vein graft atherosclerosis in apo E deficient mice.

OBJECTIVE: Use of autologous vein grafts for surgical revascularisation is limited by vein graft failure. Topical high-density lipoprotein (HDL) administration on the adventitial side of vein grafts was evaluated as a new therapeutic modality to improve vein graft patency and function. METHODS: Caval veins of C57BL/6 apo E(-/-) mice were grafted to the right carotid arteries of recipient 3 month-old C57BL/6 TIE2-LacZ/apo E(-/-) mice. HDL (200 µg/ml; 50 µl) in 20% pluronic F-127 gel was applied on the adventitial side of vein grafts. RESULTS: Topical HDL application reduced intimal area by 55% (p < 0.001) at day 28 compared to control mice. Blood flow quantified by micro magnetic resonance imaging at day 28 was 2.8-fold (p < 0.0001) higher in grafts of topical HDL treated mice than in control mice. Topical HDL potently reduced intimal inflammation and resulted in enhanced endothelial regeneration as evidenced by a 1.9-fold (p < 0.05) increase in the number of CD31 positive endothelial cells. HDL potently enhanced migration and adhesion of endothelial colony-forming cells (ECFCs) in vitro, and these effects were dependent on signaling via scavenger receptor-BI, extracellular signal-regulated kinases, and NO, and on increased ß1 integrin expression. Correspondingly, the number of CD31 ß-galactosidase double positive cells, reflecting incorporated circulating progenitor cells, was 3.9-fold (p < 0.01) higher in grafts of HDL treated mice than in control grafts. CONCLUSIONS: Topical HDL administration on the adventitial side of vein grafts attenuates vein graft atherosclerosis via increased incorporation of circulating progenitor cells in the endothelium, enhanced endothelial regeneration, and reduced intimal inflammation.

Contribution of splanchnic and musculocutaneous vascular compartments to the formation of blood flow volume in the vena cava posterior during catecholamine treatment.

Studies by electromagnetic flowmetry in acute experiments on cats under conditions of the open thoracic cage and artificial ventilation of the lungs showed that 64% of venous return via the vena cava posterior was realized at the expense of the splanchnic and 36% due to the musculocutaneous vessels (abdominal basin of the caudal vein). Epinephrine (20 µg/kg) increased the contribution of the splanchnic venous blood flow to the increase in the blood flow in the vena cava posterior and reduced the contribution of the musculocutaneous veins throughout the entire duration of systemic reactions: 84% of the blood flow increase in the vena cava posterior was due to the splanchnic and just 16% due to the musculocutaneous blood flow. Norepinephrine (10 µg/kg) resulted in a phase-wise involvement of the studied compartments in blood flow increase in the vena cava posterior. During the initial period of systemic reactions (coinciding with the maximum systemic BP rise) the contribution of the musculocutaneous compartment was 13% higher, while later (by the time of the maximum elevation of venous blood flow in the studied compartments) the contribution of splanchnic veins predominated constituting 89% of venous blood flow in the vena cava posterior. These results indicate that venous blood flow increase in the splanchnic vessels largely determined the formation of changes in the vena cava posterior blood flow in response to catecholamines.

Exercise increases the phenylephrine effects in isolated portal vein of trained rats.

Physical exercise evokes an extensive circulatory redistribution. However, the influence of exercise upon the effects of sympathomimetic agonists in veins was not well studied. Thus, the present study aimed to determine whether a single bout of exercise modifies the effects of sympathomimetic agonists in veins and whether this exercise-induced modification may be altered by exercise training. The results have shown that the training did not change the responsiveness of the rat portal vein, but exposure of trained animals to a single bout of exercise enhanced the phenylephrine Rmax in these preparations. Such exercise-induced modifications of vascular response were territory-specific since similar modifications of response to phenylephrine were not observed in vena cava. Moreover, this exercise-induced augmentation of phenylephrine Rmax in the portal vein of trained rats was prevented by endothelium removal or in the presence of N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME), indomethacin, BQ-123 or BQ-788. In conclusion, these data indicate that the training adapted the rat portal vein to respond vigorously to sympathetic stimuli even when the animal is exposed to this exercise. This increased response to sympathetic stimuli appears to involve an enhancement of the vasocontractile influence of endothelin that supplants the modulation exerted by nitric oxide (NO) and vasodilator prostanoids.

Endothelin ET(B) receptors in arteries and veins: multiple actions in the vein.

Endothelin receptors (ET(A) and ET(B)) mediate responses to ET-1. ET(B) receptor function seems to differ between a similarly sized arterial and venous pair, the rat vena cava (RVC) and rat thoracic aorta (RA). ET(B) receptors mediate RVC contraction directly, but it is unclear whether ET(B) receptors mediate contraction in RA. Because of these apparent differences in ET(B) receptor-mediated vascular contraction, we hypothesize that relaxant ET(B)-receptor mechanisms in RVC would be different from those in RA. RA and RVC rings were isolated from rats for measurement of isometric contraction. When contracted with prostaglandin F-2alpha (PGF-2alpha) (20 microM), the ET(B) receptor agonist sarafotoxin-6c (S6c) (100 nM) significantly relaxed RA and RVC. N(omega)-Nitro-L-arginine (LNNA) (100 microM) or endothelial denudation abolished relaxation to S6c in RA. By contrast, S6c-induced relaxation of RVC was attenuated but not abolished by LNNA and endothelial denudation. RVC (PGF-2alpha-contracted) relaxed to low concentrations of ET-1, whereas under the same conditions RA responded with contraction. ET-1-induced relaxation in RA was observed only with ET(A) receptor blockade. Vessels from dopamine-beta-hydroxylase-ET(B) transgenic rats, which lack functional ET(B) receptors in the vasculature, were also used. RVC (PGF-2alpha-contracted) from these rats did not relax to ET-1. Thus, although both RA and RVC possess endothelial relaxant ET(B) receptors, RA and RVC differ in that relaxant ET(B) receptors may also be present in smooth muscle of RVC. Moreover, the mechanisms of endothelial cell ET(B) receptor-mediated relaxation in RA and RVC are not the same.

Recombinant human granulocyte colony-stimulating factor enhanced the resolution of venous thrombi.

BACKGROUND: Bone marrow-derived cells are recruited into the thrombus during resolution. This study explored whether mobilization of bone marrow cells with recombinant human granulocyte colony-stimulating factor (rhG-CSF) could enhance the resolution of venous thrombi and the accumulation of macrophages in thrombi and explored the effect of rhG-CSF on cysteine-cysteine chemokine receptor 2 (CCR2) expression. METHODS: The Sprague-Dawley adult rats were randomly divided into four groups: control, sham-operated, thrombus, and treatment groups. Thrombi were induced in the thrombus and treatment group, which received a subcutaneous injection of rhG-CSF once daily for 6 days postoperatively. The thrombus, sham-operated, and control groups received equal volumes of 0.9% saline. The mononuclear cells in peripheral blood were analyzed by an automated hematology analyzer and counted under microscope. The cell marker CD68 was used to determine the number of macrophages in thrombi tissue sections. Levels of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP1alpha) in the peripheral blood were detected by enzyme-linked immunosorbent assay. Real-time reverse transcriptase-polymerase chain reaction and Western blot were used to analyze, respectively, the expression of CCR2 messenger RNA in the peripheral blood and CCR2 protein of THP-1 monocyte. RESULTS: At postoperative days 3 (P < .05) and 7 (P < .01), mononuclear cells significantly increased in treatment group (2.1 +/- 0.3, 4.4 +/- 0.3 x 10(6)/L) vs the thrombus group (1.7 +/- 0.2, 1.3 +/- 0.4 x 10(6)/L). The organization and recanalization of thrombi in treatment group progressed more quickly compared with the thrombus group (P < .01). The macrophage number of the thrombus in the treatment group (338 +/- 26 cells/15 high-power fields) increased significantly vs the thrombus group (125 +/- 11 cells/15 high-power fields, P < .01). No statistical difference was observed between the thrombus and treatment group in the MCP-1 and MIP-1alpha level in peripheral blood. Expressions of the CCR2 gene in the peripheral blood of the treatment group significantly increased compared with the thrombus group (P < .05). Recombinant human G-CSF induced higher expression of CCR2 protein of human monocytic cell line THP-1. CONCLUSIONS: Bone marrow mobilization enhanced the resolution and recanalization of venous thrombi. This process was associated with increased macrophage accumulation in thrombi, which might be the result of higher CCR2 expression of monocytes. CLINICAL RELEVANCE: The classic treatment of venous thrombi is anticoagulation. Anticoagulant therapy and thrombolysis both have limited effects on existing thrombi and have a small but significant risk of severe hemorrhage. In clinical practice, we lack specific treatment for patients with venous thrombosis combined with brain hemorrhage or a gastrointestinal activated ulcer, which are contraindicated for anticoagulation and thrombolytic therapy. Enhancing the resolution of venous thrombi would contribute to its therapy. Bone marrow-derived cells are recruited into the thrombus during resolution. Many of these cells express a macrophage phenotype and may represent a population of plastic stem cells that orchestrate thrombus recanalization. Recombinant human granulocyte colony stimulating factor (rhG-CSF) can mobilize monocytic lineage cells into peripheral blood and may contribute to this cell in the thrombi. If rhG-CSF enhances the resolution of venous thrombi and recanalization, it might be used to treat patients with venous thrombi, especially those who have contraindication for anticoagulation and thrombolytic therapy.

Morphological and biochemical characterization of remodeling in aorta and vena cava of DOCA-salt hypertensive rats.

Arterial remodeling occurs in response to mechanical and neurohumoral stimuli. We hypothesized that veins, which are not exposed to higher pressures in hypertension, would demonstrate less active remodeling than arteries. We assessed remodeling with two standard measures of arterial remodeling: vessel morphometry and the expression/function of matrix metalloproteinases (MMPs). Thoracic aorta and vena cava from sham normotensive and DOCA-salt hypertensive rats (110 +/- 4 and 188 +/- 8 mmHg systolic blood pressure, respectively) were used. Wall thickness was increased in DOCA-salt vs. sham aorta (301 +/- 23 vs. 218 +/- 14 mum, P < 0.05), as was medial area, but neither measure was altered in the vena cava. The aorta and vena cava expressed the gelatinases MMP-2, MMP-9, transmembrane proteinase MT1-MMP, and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemically, MMP-2 localized to smooth muscle in the aorta and densely in endothelium/smooth muscle of the vena cava. Western and zymographic analyses verified that MMP-2 was active in all vessels and less active in the vena cava than aorta. In hypertension, MMP-2 expression and activity in the aorta were increased (59.1 +/- 3.7 and 74.5 +/- 6.1 units in sham and DOCA, respectively, P < 0.05); similar elevations were not observed in the vena cava. MMP-9 was weakly expressed in all vessels. MT1-MMP was expressed by the aorta and vena cava and elevated in the vena cava from DOCA-salt rats. TIMP-2 expression was significantly increased in the aorta of DOCA rats compared with sham but was barely detectable in the vena cava of sham or DOCA-salt hypertensive rats. These findings suggest that large veins may not undergo vascular remodeling in DOCA-salt hypertension.

Cyclooxygenase, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK, Rho kinase, and Src mediate hydrogen peroxide-induced contraction of rat thoracic aorta and vena cava.

In hypertension, blood vessels exhibit increased reactive oxygen species production that may alter vascular tone. We previously observed that H2O2 contracted rat thoracic vena cava under resting tone and aorta contracted with KCl. In arteries but not veins, H2O2-induced contraction required extracellular Ca2+ influx. Because of this difference in Ca2+ utilization, we hypothesized that signaling pathways mediating H2O2-induced contraction in vena cava under resting tone differed from those mediating H2O2-induced contraction in aorta contracted with KCl. Inhibitors of cyclooxygenase (COX) 1 and 2 (indomethacin, 10 microM), thromboxane A2 (TXA2) receptors [ICI185282 (2RS,4RS,5SR-4-o-hydroxyphenyl-2-trifluoromethyl-1,3-dioxan-5-yl heptenoic acid), 10 microM], p38 mitogen-activated protein kinase (MAPK) [SB203580 (4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine), 10 microM], extracellular signal-regulated kinase (Erk) [PD98059 (2'-amino-3'-methoxyflavone), 10 microM], src [PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, 10 microM], and rho kinase [Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), 10 microM], significantly reduced H2O2-induced contraction in vena cava under resting tone and aorta after KCl (30 mM) contraction. In contrast, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, 20 microM] did not reduce aortic or venous H2O2-induced contraction. p38 MAPK, Erk MAPK, and src inhibition did not reduce aortic or venous contraction to the TXA2 receptor agonist U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy PGF(2alpha), 1 microM), whereas rho kinase inhibition significantly reduced aortic and venous contraction to U46619, and PI3-K inhibition reduced venous contraction to U46619. Our data suggest that, in rat thoracic aorta and vena cava, a COX-derived metabolite is one important mediator of H2O2 contraction, possibly via rho kinase activation, and that H2O2-induced contraction via p38 and Erk MAPK probably occurs independently of TXA2 receptor activation.

Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. Since the role of TAFI in thrombus formation is still controversial in mice, our present study was designed to evaluate mice deficient in TAFI (TAFI(-/-)) on FeCl(3)-induced vena cava and carotid artery thrombosis. Parallel studies were carried out in wild-type mice using a potato carboxypeptidase inhibitor (PCI), a selective inhibitor of activated TAFI (TAFIa). Significant reduction in thrombus formation was observed in TAFI(-/-) mice (n = 8, P < 0.05 compared to wild-type littermates) but not in heterozygous (TAFI(+/-)) mice in 3.5% FeCl(3)-induced vena cava thrombosis. A similar effect was observed following treatment with 5 mg/kg bolus plus 5 mg/kg/h PCI in the same venous thrombosis model in C57BL/6 mice (n = 8, P < 0.01 compared to vehicle). No compositional difference was observed for the venous thrombi in TAFI(-/-) and wild-type littermates with or without PCI treatment using histological assessment. In contrast, neither TAFI deficiency nor treatment with PCI showed antithrombotic efficacy in the 3.5% FeCl(3)-induced carotid artery thrombosis model. In a tail transection bleeding time model, both TAFI deficiency and PCI treatment increased bleeding time up to 4.5 and 3.5 times, respectively, over controls (P < 0.05, n = 8). Similar ex vivo fibrinolytic activities were demonstrated for both TAFI deficiency and PCI treatment as enhanced lysis of thrombin-induced plasma clots and lysis of whole blood clot in a thrombelastograph. These data provide direct evidence for the role of TAFIa in vena cava thrombosis without the addition of exogenous thrombolytic in mice. The strong ex vivo fibrinolytic activity of TAFI deficiency or TAFIa inhibition by PCI provides a biomarker of TAFIa inhibition that tracks in vivo antithrombotic efficacy.

Pleiotropic effects of hydrogen peroxide in arteries and veins from normotensive and hypertensive rats.

Hydrogen peroxide causes vascular contraction and relaxation and contributes to the pathogenesis of hypertension. We hypothesized that the contractile state of blood vessels governs whether H2O2 causes contraction or relaxation. Hydrogen peroxide (1 micromol/L to 1 mmol/L) concentration-dependently contracted thoracic aorta and vena cava from sham normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The maximal contraction to H2O2 was 3 times greater in DOCA aorta compared with sham aorta but unchanged in DOCA vena cava compared with sham vena cava. In prostaglandin F2alpha (20 micromol/L)-contracted aorta and vena cava from sham and DOCA rats, H2O2 (1 micromol/L to 1 mmol/L) induced a concentration-dependent relaxation that was impaired in DOCA aorta but not DOCA vena cava. In contrast, in KCl (30 mmol/L)-contracted vessels, maximal H2O2-induced contraction was enhanced 15-fold in sham aorta and 5-fold in DOCA aorta but only 2-fold in sham vena cava. Tetraethylammonium (10 mmol/L), BAY K 8644 (100 nmol/L), and ouabain (1 mmol/L) all enhanced maximal aortic H2O2-induced contraction, whereas only ouabain enhanced venous H2O2-induced contraction. The removal of extracellular Ca2+ reduced H2O2-induced contraction in KCl-contracted aorta, whereas maximal venous H2O2-induced contraction (under basal conditions) was unchanged. Our data suggest that differences in arterial and venous K+ channel activity and extracellular Ca2+ influx are responsible for differences in arterial and venous contraction to H2O2. In DOCA-salt hypertension, arterial but not venous contraction to H2O2 is enhanced, and relaxation to H2O2 is reduced.

Tissue-specific modulation of cyclooxygenase-2 (Cox-2) expression in the uterus and the v. cava by estrogens and phytoestrogens.

Cyclooxygenase 2 (Cox-2), an enzyme involved in prostaglandin production, is a key player in the development of pathologic changes, such as colorectal cancer, arteriosclerosis and thrombosis. In this study, we investigated the effects of estrogens, selective estrogen receptor modulators (SERMs), pure antiestrogens and phytoestrogens on the tissue-specific expression of Cox-2 in the uterus and the v. cava of ovariectomized female rats. Cox-2 expression could be detected in the uterine epithelium and in the endothelium of the v. cava. Cox-2 expression was time-dependently stimulated after administration of 17beta estradiol (E2) in the uterus. In the v. cava, E2 treatment resulted in a stimulated expression of the progesterone receptor (PR), a gene known to be regulated by E2, whereas Cox-2 was simultaneously down-regulated. Administration of the pure antiestrogen faslodex (Fas) had no effect on Cox-2 expression. In contrast, administration of tamoxifen (Tam) resulted in a decrease of Cox-2 expression in the v. cava but does not stimulate Cox-2 expression in the uterus. Interestingly, the same expression pattern of Cox-2 could be detected after dose-dependent administration of genistein (Gen). Here, down-regulation of Cox-2 could already be detected after administration of merely 0.5 mg/(kgBW) Gen, a dose where no effects on uterine weight were observed. In summary, our results demonstrate a reverse tissue-specific regulation of Cox-2 expression by estrogens in the v. cava and uterus indicating the existence of complex molecular mechanisms which have to be characterized in future studies. Remarkably, Tam and the phytoestrogen Gen, both share the ability to decrease the expression of Cox-2 in the v. cava without effecting its uterine expression. These observations may be of great importance with respect to potential beneficial or adverse effects of estrogens, SERMs and phytoestrogens on the cardiovascular tissue.

No estrogen-like effects of an isopropanolic extract of Rhizoma Cimicifugae racemosae on uterus and vena cava of rats after 17 day treatment.

The effects of black cohosh extracts (Rhizoma Cimicifugae racemosae) on primary estrogen target organs, like mammary gland and endometrium are better described then those on other estrogen-sensitive systems e.g. the vasculature. We therefore treated ovariectomized DA/Han rats for 17 days with an isopropanolic Cimicifuga racemosa rhizoma extract (iCR) alone and in combination with the pure antiestrogen fulvestrant. As control groups vehicle, estradiol, fulvestrant, and estradiol fulvestrant cotreatment were used. Effects of all substances were investigated by vena cava and uterine gene expression analysis using real-time-PCR. Uterus wet weight was increased after estradiol treatment compared to the negative controls but none of the other treatments including the treatment with iCR had a uterotrophic effect. While estradiol-induced changes in uterine gene expression were mainly analogous to those detectable in shorter term experiments, iCR showed no or slightly antiestrogenic effects on gene expression in the uterus. This is mirrored in the vena cava where iCR had a very minor impact on the expression of the genes analyzed. While C. racemosa is effectively used for treatment of peri- and post-menopausal symptoms for a long time its mechanism of action remains unresolved. Contrary to earlier suggestions C. racemosa does not seem to act as an estrogen agonist, but possibly as a weak antiestrogen.

Inhibitory effects of ethanol extract from Radix Ophiopogon japonicus on venous thrombosis linked with its endothelium-protective and anti-adhesive activities.

The in-vivo inhibitory effects of the ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) on venous thrombosis were studied in mouse and rat models and in-vitro endothelial cell-protective and anti-adhesive activities were observed in ECV304 cells injured by sodium dithionite and HL-60 adhesion to ECV304 cells injured by TNF-alpha. The in-vivo results showed that ROJ-ext significantly inhibited venous thrombosis induced by tight ligation of the inferior vena cava for 6 h in mice and for 24 h in rats by once oral administration at doses of 12.5 and 25 mg/kg. Meanwhile, ROJ-ext had no obvious effect on some coagulation parameters, which was different from warfarin, which remarkably prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) in rats at the same time. Histological analysis under light microscope and scanning electron microscope (SEM) of inferior vena cava indicated that ROJ-ext could protect endothelial cells from anoxic injury and alleviate inflammatory changes in the vein wall. On the other hand, the in-vitro studies approved that ROJ-ext significantly enhanced viability of ECV304 cells injured by sodium dithionite at the concentrations of 0.1, 1.0 and 10 mug/ml when given before and after the anoxic induction. Meanwhile, ROJ-ext remarkably inhibited adhesion of HL-60 cells to ECV304 cells injured by rh TNF-alpha at above concentrations in a dose-dependent manner. The findings of this study showed that ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) inhibited venous thrombosis, which linked with its endothelial cell-protective and anti-adhesive activities. This lends scientific support to the therapeutic use of the plant for thrombotic diseases.

Nitric oxide control of large veins in the toad Bufo marinus.

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10(-5) M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10(-5) M). The NOS inhibitors, L-NNA (10(-4) M) and L-NAME (10(-4) M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10(-7)-10(-8) M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10(-5) M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.