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.

In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor.

BACKGROUND: Smooth muscle cell (SMC) migration and proliferation are important in the development of intimal hyperplasia, the major cause of vein graft failure. Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system are pivotal in extracellular matrix degradation and, by that, SMC migration. Previously, we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the receptor-binding amino terminal fragment (ATF) of urokinase, reduces SMC migration and neointima formation in an in vitro restenosis model using human saphenous vein cultures more efficiently than both protease systems separately. Because use of viral gene delivery is difficult in clinical application, this study used nonviral delivery of TIMP-1.ATF plasmid to reduce vein graft disease in a murine bypass model. Nonviral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery, such as immune responses and short-term expression. METHODS: Plasmids encoding ATF, TIMP-1, TIMP-1.ATF, or luciferase, as a control, were injected and electroporated in both calf muscles of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3Leiden) mice (n = 8). One day after electroporation, a venous interposition of a donor mouse was placed into the carotid artery of a recipient mouse. In this model, vein graft thickening develops with features of accelerated atherosclerosis. Vein grafts were harvested 4 weeks after electroporation and surgery, and histologic analysis of the vessel wall was performed. RESULTS: Electroporation-mediated overexpression of the plasmid vectors resulted in a prolonged expression of the transgenes and resulted in a significant reduction of vein graft thickening (ATF: 36% +/- 9%, TIMP-1: 49% +/- 5%, TIMP-1.ATF: 58% +/- 5%; P < .025). Although all constructs reduced vein graft thickening compared with the controls, the luminal area was best preserved in the TIMP-1.ATF-treated mice. CONCLUSION: Intramuscular electroporation of TIMP-1.ATF inhibits vein graft thickening in vein grafts in carotid arteries of hypercholesterolemic mice. Binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface enhances the inhibitory effect of TIMP-1 on vein graft remodeling in vitro as well as in vivo and may be an effective strategy to prevent vein graft disease.

Effect of matrix metalloproteinase-9 knockout on vein graft remodelling in mice.

Long-term success in vein grafting for bypassing arteries blocked by atherosclerosis is limited by migration and proliferation of smooth muscle cells to form a neointima. Matrix metalloproteinases (MMPs), in particular MMP-2 and MMP-9, are implicated in neointimal formation by freeing smooth muscle cells from the cell-matrix contacts that normally restrict migration. We investigated the role of MMP-9 in vein grafts directly, using knockout mice. Vein grafts in MMP-9(-/-) and wild-type mice had similar luminal and graft areas at 1, 4 and 8 weeks after engraftment, increasing with time. There was a relationship between the perimeter of the external elastic lamina and graft thickness (indicating graft remodelling) in MMP-9(-/-) mice at 1 week after surgery not apparent in control mice until later (r(2) = 0.933 for MMP-9(-/-) mice, r(2) = 0.040 for wild-type mice). Grafts in MMP-9(-/-) mice had 6-fold more pro- and active MMP-2 (p = 0.013, p = 0.026) than grafts in wild-type mice. Grafts from MMP-9(-/-) mice also had more collagen (p = 0.046 at 8 weeks), without any difference in cell number. Thus, while a lack of MMP-9 did not alter vein graft wall area or cellularity, grafts from MMP-9(-/-)mice accumulated more collagen and had earlier linear expansive remodelling, possibly due to an early compensatory increase in MMP-2.

A comparison of reactive oxygen species metabolism in the rat aorta and vena cava: focus on xanthine oxidase.

Reactive oxygen species (ROS) are important mediators in vascular biology. Venous function, although relevant to cardiovascular disease, is still understudied. We compared aspects of ROS metabolism between a major artery (the aorta) and a major vein (the vena cava, VC) of the rat, with the hypothesis that venous ROS metabolism would be overall increased compared with its arterial counterpart. Superoxide and hydrogen peroxide (H2O2) release in basal conditions was higher in VC compared with aorta. The antioxidant capacity for H2O2 was also higher in VC than in aorta. Exogenous superoxide induced a higher contraction in VC compared with aorta. Protein expression of three major ROS metabolizing enzymes, xanthine oxidase (XO), CuZn-SOD, and catalase, was higher in VC compared with aorta. Because XO seemed a likely source of the higher VC ROS levels, we examined it further and found higher mRNA expression and activity of XO in VC compared with aorta. We also investigated the impact of XO inhibition by allopurinol on aorta and VC functional responses to norepinephrine, ANG II, ET-1, and ACh. Maximal ET-1-mediated contraction was decreased by allopurinol in VC but not in the aorta. Our results suggest that there are overall differences in ROS metabolism between aorta and VC, with the latter operating normally at a higher set point, releasing but also being able to handle, higher ROS levels. We propose XO to be an important source for these differences. The result of this particular comparison may be reflective of a general arteriovenous contrast.

Variable expression of 11beta Hydroxysteroid dehydrogenase (11beta-HSD) isoforms in vascular endothelial cells.

Vascular tissue expresses two isoforms of the enzyme 11beta-Hydroxysteroid dehydrogenase, 11beta-HSD1 and 11beta-HSD2. These enzymes are responsible for the local metabolism of endogenous glucocorticoids (GCs). 11beta-HSD1 deactivates GCs to their 11keto metabolites or transforms inert 11keto metabolites back to active GCs. Although, bi-directional, vascular 11beta-HSD1 favors reactivation (reductase) over the deactivation (dehydrogenase) reaction, 11beta-HSD2 only functions as a dehydrogenase. GC deactivation by enhanced 11beta-HSD2 dehydrogenase activity or by impaired 11beta-HSD1 reductase activity correlates with lower vascular resistance. These studies were designed to demonstrate the existence and regulation of these isoforms in vascular endothelial cells and to determine whether the expression varied by species and locale. Western blots were prepared from pre-confluent and confluent cultures of human umbilical vein endothelial cells (HUVEC). 11beta-HSD1 was clearly expressed while 11beta-HSD2 was much less prominent. Cultured rat aortic and bovine glomerular endothelial cells showed a similar pattern. Using immunohistochemistry, endothelial cells from human and mouse artery preparations clearly demonstrated 11beta-HSD1. In separate experiments, pre-confluent growing HUVEC expressed more 11beta-HSD1 compared to confluent cells. Serum-deprived growth-retarded HUVEC expressed significantly less 11beta-HSD1. The enhanced expression of 11beta-HSD1 was also observed 24h following a scratch "injury" to the culture plates. Changes in 11beta-HSD1 with growth and during repair occurred at the transcription level. Thus, 11beta-HSD1 protein expression predominates in endothelial cells and varies during periods of growth.

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.

Nitric oxide synthase activity in hyperthyroid and hypothyroid rats.

OBJECTIVE: Thyroid disorders are accompanied by important changes in haemodynamic and cardiac functions and renal sodium handling. Since nitric oxide (NO) plays a crucial role in regulating vascular tone and renal sodium excretion, the present paper was designed to determine whether changes in the activity of NO synthase (NOS) participate in the cardiovascular and renal manifestations of thyroid disorders. METHODS: We measured NOS activity in the heart (left and right ventricles), vessels (aorta and cava) and kidney (cortex and medulla) of euthyroid, hyperthyroid and hypothyroid rats after 6 weeks of treatment. NOS activity was determined by measuring the conversion of L-[(3)H]-arginine to L-[(3)H]-citruline. RESULTS: NOS activity was higher in all tissues from hyperthyroid rats when compared with controls, except in the right ventricle. In the hypothyroid group, NOS activity showed a more heterogeneous pattern, with significant increases in both ventricles but significant reduction in the aorta, while in the vena cava, renal cortex and medulla the enzyme activity also tended to be higher, but significance was not reached. CONCLUSIONS: These data indicated that NOS activity was upregulated in tissues primarily related to blood pressure control in hyperthyroid rats, suggesting that an increased NO production may contribute to the hyperdynamic circulation in hyperthyroidism and may have a protective homeostatic effect in the target organs of the hypertension that accompanies this endocrine disease. The aortic and renal findings in hypothyroid rats suggested a possible role for NOS in the increased peripheral resistance and the normal pressure-diuresis-natriuresis response of these hypotensive animals, although hypothyroidism produced a heterogeneous tissue response in NOS activity.

[Intensity of lipid peroxidation and antioxidant enzyme activity in arterial and venous walls during hypervitaminosis D]. / Intensyvnist' protsesiv perekysnoho okysnennia lipidiv ta antyoksydantna aktyvnist' arterial'noï i venoznoï stinky v dynamitsi rozvytku hipervitaminozu D.

The intensity of the lipid peroxydation (LPO) and the antioxidant enzyme activity (superoxide dismutase, glutathione peroxydase and catalase) on injecting vitamin D in high doses (10,000 U/kg) was examined in the arterial and venous walls of rabbits. The increase in the amount of the intermediate and final LPO products has been found in the vessels of all types. The lowest intensity of LPO was noted in the vena cava. The decrease in the antioxidant activity has been revealed. But vena cava inferior was the exception because the activity of all studied antioxidant enzymes grew in its wall. This increase is likely to be one of the reasons for vena resistance to the action of damaging factors.

Haem oxygenase activity in human umbilical cord and rat vascular tissues.

Carbon monoxide (CO) has been shown to affect vascular tone in smooth muscle cells and thus, may regulate regional or systemic blood pressure as well as fetoplacental vascular tone and fetal blood delivery. To assess the potential of vascular tissue to produce CO, we determined haem oxygenase (HO) activity through in vitro quantitation of CO production with gas chromatography and its inhibition by 33-66 microm of chromium mesoporphyrin (CrMP) in homogenate preparations of rat aorta and vena cava and human umbilical cord tissues. We compared these results to HO activity in rat heart and liver. We also discuss normalization of HO activity on a per mg protein as well as per g fresh weight (FW) tissue basis. We found that both rat vascular tissue HO activities (per g FW) were equal, but greater than that of heart (x3) and less than that of liver (x0.2). For human cord tissues, HO activities of artery and vein were equal, but greater than that of Wharton's jelly. Also, HO activity in rat vascular tissues was 3x greater than that of the human cord tissues. HO activity was completely inhibited by CrMP in rat heart (90 per cent) and liver (96 per cent), but incompletely (50-66 per cent) in both rat and human vascular tissues. We established that it is unlikely that other non-haem CO-generating processes account for this unique insensitivity of HO to CrMP inhibition. In fact, high concentrations of other potent metalloporphyrin inhibitors affected vascular tissue HO even less. We found that the degree of in vitro HO inhibition appeared to be related to the concentration of haem in the reaction medium. We conclude that the presence of HO activity in cord tissues supports the possibility that CO plays a role in fetoplacental blood flow regulation.

Cellular and subcellular localization of catalase in the heart of transgenic mice.

Previous studies have described a cardiac-specific, catalase-overexpressing transgenic mouse model that was used to study myocardial oxidative injury. This study was undertaken to demonstrate cellular and subcellular localization of catalase in the hearts of transgenic mice. By the light microscopic immunoperoxidase method, we found that the overexpressed catalase was exclusively localized in cardiomyocytes. The ratios of immunoreactive cardiomyocytes in the heart were quite different among three transgenic lines examined but agreed with the elevated levels of catalase activity. In the cardiac blood vessels, positive cells were found in the walls of pulmonary veins and the vena cava, which consist of cardiomyocytes, but not in the pulmonary arteries, aorta, or cardiac valves. The electron microscopic immunogold method revealed that the elevated catalase was in sarcoplasm, nucleus, and peroxisomes, but not in mitochondria. In contrast to these distributions, catalase in the non-transgenic cardiomyocytes was in peroxisomes only. In addition, the number and size of peroxisomes in the transgenic cardiomyocytes were markedly increased, but no other ultrastructural changes were observed in comparison with those of non-transgenic mice. These results demonstrated that the elevated catalase in transgenic mouse heart is localized in cardiomyocytes and is distributed to peroxisomal and extraperoxisomal, but not mitochondrial, compartments.

Aromatase and sex steroid receptors in human vena cava.

Among sex steroids, especially estrogen metabolism has been considered to play a role in the function and pathology of human veins. We investigated the expression and activity of the estrogen-producing enzyme aromatase and estrogen receptor (ER) in human vena cava to assess possible in situ biosynthesis of estrogens and their modes of action. We first examined aromatase expression by immunohistochemistry in human inferior vena cava obtained from 29 autopsy cases (11 males, 18 females, 63.6 +/- 3.0 years old). We then semiquantitated the level of aromatase mRNA by reverse transcriptase-polymerase chain reaction in 24 cases and aromatase activity by 3H-water assay in 15 cases to examine whether or not and in which cell types aromatase was expressed. We also studied alternative use of multiple exon 1s of its gene and immunolocalization of 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD I), which converts estrone produced by aromatase to estradiol, a biologically active estrogen and ER. Aromatase and 17beta-HSD I immunoreactivity were both detected in smooth muscle cells (SMC) of the media in all the cases and in endothelial cells (EC) in 20 and 22 cases, respectively. ER immunoreactivity was detected in SMC of vena cava in 21 cases. The amount of aromatase mRNA was significantly greater in the cases utilizing 1c (I.3) or 1d (P.II) of exon 1 (9 cases, 191.1 +/- 26.3 attomol/ng total RNA) than those utilizing 1b (I.4) as the promoter (14 cases, 50.6 +/- 13.0 attomol/ng total RNA) (p < 0.01). Significant correlation (p < 0.05) was observed between the amount of aromatase mRNA and aromatase activity in 15 cases examined. No significant correlation was detected between the amount of aromatase mRNA or aromatase labeling index and the ER status. These results suggest that estrone and estradiol are produced in the human vena cava and that their production is mediated by aromatase and 17beta-HSD I, respectively but not all of these locally synthesized estrogens may not work directly in situ.

Intermediate filaments and ATPase activity in the vascular wall of vertebrates.

The vascular wall of aorta and vena cava was examined for adenosine triphosphatase (ATPase) activity and cytoskeletal intermediate filaments (IF) in different representatives of vertebrates. Enzyme activity was studied by the modified method of Padykula and Herman. A streptavididin-biotin immunohistochemical method was applied to reveal desmin (D) and vimentin (V) IF. Endothelial cells of all vessels were V-positive and D-negative and exhibit high ATPase activity. Vascular smooth muscle cells (SMC) in lower vertebrates (pisces and amphibia) were also V-positive and D-negative, but showed low ATPase activity. SMC were D-positive and V-negative and possessed high enzyme activity in aves and mammals, similar to that of the endothelium. In cow vascular wall D-reactivity and high ATPase activity were mostly expressed in bundles of mosaically arranged thick SMC fibres of the outer aortic media as well as in the longitudinal fibres in the inferior vena cava. In higher vertebrates SMC of vasa vasorum were both V- and D-positive and showed high enzyme activity. The results demonstrate that D-immunoreactivity is mostly expressed in SMC of layers of high functional activity, which correlates with the intense ATPase reaction in these cells.

Evidence for a potent angiotensin I degrading enzyme different from angiotensin I converting enzyme in rat vascular tissues.

There are indications for the existence of an intrinsic renin angiotensin system in vascular walls, which is assumed to participate in blood pressure regulation and in pathogenesis of arterial hypertension. It was evaluated if and to what extent the decapeptide angiotensin (A) I, one of the natural substrates of A I converting enzyme (ACE), is degraded by other peptidases than ACE in rat vascular tissues. A I and A II degradation was studied in arterial and venous vascular wall extracts. The activities ranged between 0.068 +/- 0.025 U and 0.044 +/- 0.025 U. The enzymes involved were biochemically characterized by determination of isoelectric points (pI), pH optima, molecular weights and by investigation of their inhibition behavior in vitro. One potent A I degrading enzyme (AIDE) was identified with pI between 3.6 and 3.9, and pH optimum at 7.75. In vitro studies revealed that AIDE activity was not blocked by the specific ACE inhibitors MK 421 or MK 422 (both 11 nMol/ml). The molecular weight of AIDE ranged between 440,000 and 457,000. The results indicate that AIDE is not identical to ACE (pI 4.2-5.0; pH optimum 8.3). AIDE was also observed in aortic smooth muscle cells cultured in vitro. AIDE decreased following bilateral nephrectomy or administration of aldosterone combined with sodium chloride loading, whereas it was elevated in spontaneously hypertensive rats (Okamoto strain). Since AIDE metabolizes A I, one of the substrates of ACE, it may indirectly affect A II formation and bradykinin inactivation as well.

Specific cerebrovascular localization of glutamate decarboxylase activity.

Glutamate decarboxylase (GAD) activity and GABA levels, determined with a [3H]muscimol radioreceptor assay, were found to be significantly higher in cerebral blood vessels from the piaarachnoid membrane as compared to extracranial vessels (aorta, mesenteric and femoral arteries, and vena cava). A cerebrovascular localization for GABA and GAD is consistent with earlier studies suggesting that an indigenous GABA system is involved in cerebral vascular function.