Vascular Tissue Levels Research Articles

Modulators of the Vascular Endothelin Receptor in Blood Pressure Regulation and Hypertension

Endothelin (ET) is one of the most investigated molecules in vascular biology. Since its discovery two decades ago, several ET isoforms, receptors, signaling pathways, agonists and antagonists have been identified. ET functions as a potent endothelium-derived vasoconstrictor, but could also play a role in vascular relaxation. In endothelial cells, preproET and big ET are cleaved by ET converting enzymes into ET-1, -2, -3 and -4. These ET isoforms bind with different affinities to ET(A) and ET(B) receptors in vascular smooth muscle (VSM), and in turn increase [Ca(2+)](i), protein kinase C and mitogen-activated protein kinase and other signaling pathways of VSM contraction and cell proliferation. ET also binds to endothelial ET(B) receptors and stimulates the release of nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor. ET, via endothelial ET(B) receptor, could also promote ET re-uptake and clearance. While the effects of ET on vascular reactivity and growth have been thoroughly examined, its role in the regulation of blood pressure and the pathogenesis of hypertension is not clearly established. Elevated plasma and vascular tissue levels of ET have been identified in salt-sensitive hypertension and in moderate to severe hypertension, and ET receptor antagonists have been shown to reduce blood pressure to variable extents in these forms of hypertension. The development of new pharmacological and genetic tools could lead to more effective and specific modulators of the vascular ET system for treatment of hypertension and related cardiovascular disease.

Surgical and histopathological effects of topical Ankaferd® hemostat on major arterial vessel injury related to elevated intra-arterial blood pressure.

The aim of this study was to assess the surgical and histopathological hemostatic effects of topical Ankaferd blood stopper (ABS) on major arterial vessel injury related to elevated intra-arterial blood pressure in an experimental rabbit model. The study included 14 New Zealand rabbits. ABS was used to treat femoral artery puncture on 1 side in each animal and the other untreated side served as the control. Likewise, for abdominal aortic puncture, only 50% of the aortic injuries received topical liquid ABS and the others did not (control). The experiment was performed under conditions of normal arterial blood pressure and was repeated with a 50% increase in blood pressure. Histopathological analysis was performed in all of the studied animals. Mean bleeding time in the control femoral arteries was 105.0±18.3 s, versus 51.4±9.8 s (p<0.05) in those treated with ABS. Mean blood loss from the punctured control femoral arteries was 5.0±1.5 mg and 1.6±0.4 mg from those treated with ABS (p<0.05). Histopathological examination of the damaged arterial structures showed that ABS induced red blood cell aggregates. ABS administered to experimental major arterial vessel injury reduced both bleeding time and blood loss under conditions of normal and elevated intra-arterial blood pressure. ABS-induced erythroid aggregation was prominent at the vascular tissue level. These findings will inform the design of future experimental and clinical studies on the anti-bleeding and vascular repairing effects of the novel hemostatic agent ABS.

Novel mutations in TTC37 associated with tricho-hepato-enteric syndrome

The Tricho-Hepato-Enteric (THE) syndrome is an autosomal recessive condition marked by early and intractable diarrhea, hair abnormalities, and immune defects. Mutations in TTC37, which encodes the putative protein Thespin, have recently been associated with THE syndrome. In this article, we extend the pattern of TTC37 mutations by the description of 11 novel mutations in 9 patients with a typical THE syndrome. The mutations were spread along the gene sequence, none of themrecurrent. Different types of mutation were observed: frameshift mutations, splice-site altering mutations, or missense mutations, most of them leading to the creation of a premature stop codon. Concurrently, we investigated the pattern of TTC37 expression in a panel of normal human tissues and showed that this gene is widely expressed, with high levels in vascular tissues, lymph node, pituitary, lung, and intestine. In contrast, TTC37 is not expressed in the liver, an organ that is not consistently affected in THE syndrome. Last, we suggested a model for the putative structure of the unknown Thespin protein.

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz)

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

Effect of Multiparity on Vascular Compliance and Collagen Content

We have shown that vascular compliance is decreased in multiparous rats. When the balance of collagen synthesis/breakdown changes, the functional characteristics of the vessels are altered. Collagen degradation is regulated by matrix metalloproteinases (MMPs). We have previously shown that multiparity increases oxidative stress and peroxynitrite formation. Peroxynitrite can change MMP levels in vascular tissues and thus alter collagen content. We examined the effect of multiparity on collagen content of aorta and mesenteric arteries by Trichrome Masson staining of paraffin imbedded vascular sections, by total vascular collagen and by hydroxyproline. MMP2 activity in vascular tissues was measured by gelatin zymography. Functional changes in vascular compliance were assessed by pressure myography. Data demonstrate that multiparity is associated with decreased MMP2 activity in aorta (870±229 vs 4797±1116) and mesenteric arteries (544±113 vs 2294±777). This was accompanied by increased total collagen (aorta: 536±37 vs 259±32; mesentery: 426±24 vs 206±12) and hydroxyproline (aorta: 149.5±3 vs 64±7; mesentery: 142.8±12.8 vs 74.0±11). Vascular compliance was decreased in vessels from multiparous rats. These data suggest that multiparity decreases vascular compliance via mechanisms involving MMP‐regulated collagen degradation which is downstream of the peroxynitrite signalling pathway.

Carbohydrate-related genes and cell wall biosynthesis in vascular tissues of loblolly pine (Pinus taeda)

Loblolly pine (Pinus taeda L.), the most widely planted tree species in the United States, is an important source of wood and wood fibers for a multitude of consumer products. Wood fibers are primarily composed of secondary cell walls, and cellulose, hemicelluloses and lignin are major components of wood. Fiber morphology and cell wall composition are important determinants of wood properties. We used comparative genomics to identify putative genes for cellulose and hemicellulose synthesis in loblolly pine that are homologous to genes implicated in cell wall synthesis in angiosperms. Sequences encoding putative secondary cell wall cellulose synthase genes, cellulose synthase-like genes, a membrane-bound endoglucanase gene, a sucrose synthase gene, a UDP-glucose pyrophosphorylase gene and GDP-mannose pyrophosphorylase genes were identified in expressed sequence tag (EST) collections from loblolly pine. Full-length coding sequences were obtained from cDNA clones isolated from a library constructed from developing xylem. Phylogenetic relationships between the genes from loblolly pine and angiosperm taxa were examined and transcriptional profiling in vascular tissues was conducted by real-time quantitative, reverse transcriptase-polymerase chain reaction. The putative cell wall synthesis genes were expressed at high levels in vascular tissues and a subset was differentially regulated in xylem and phloem tissues. Inferred phylogenetic relationships and expression patterns for the genes from loblolly pine were consistent with roles in synthesis of complex carbohydrates of the cell wall. These studies suggest functional conservation of homologous wood formation genes in gymnosperm and angiosperm taxa.

Global Improvement of Vascular Function and Redox State With Low-Dose Folic Acid

Although dietary folate fortification lowers plasma homocysteine and may reduce cardiovascular risk, high-dose folic acid therapy appears to not alter clinical outcome. Folic acid and its principal circulating metabolite, 5-methyltetrahydrofolate, improve vascular function, but mechanisms relating folate dose to vascular function remain unclear. We compared the effects of folic acid on human vessels using pharmacological high-dose versus low-dose treatment, equivalent to dietary folate fortification. Fifty-six non-folate-fortified patients with coronary artery disease were randomized to receive low-dose (400 microg/d) or high-dose (5 mg/d) folic acid or placebo for 7 weeks before coronary artery bypass grafting. Vascular function was quantified by magnetic resonance imaging before and after treatment. Vascular superoxide and nitric oxide bioavailability were determined in segments of saphenous vein and internal mammary artery. Low-dose folic acid increased nitric oxide-mediated endothelium-dependent vasomotor responses, reduced vascular superoxide production, and improved enzymatic coupling of endothelial nitric oxide synthase through availability of the cofactor tetrahydrobiopterin. No further improvement in these parameters occurred with high-dose compared with low-dose treatment. Whereas plasma 5-methyltetrahydrofolate increased proportionately with treatment dose of folic acid, vascular tissue 5-methyltetrahydrofolate showed no further increment with high-dose compared with low-dose folic acid. Low-dose folic acid treatment, comparable to daily intake and dietary fortification, improves vascular function through effects on endothelial nitric oxide synthase and vascular oxidative stress. High-dose folic acid treatment provides no additional benefit. These direct vascular effects are related to vascular tissue levels of 5-methyltetrahydrofolate rather than plasma levels. High-dose folic acid treatment likely confers no further benefit in subjects already receiving folate supplementation.

Cytochemical immuno-localization of allene oxide cyclase, a jasmonic acid biosynthetic enzyme, in developing potato stolons

The involvement of jasmonates in the tuber development has been proved by the presence of many of these compounds in potato stolons, modification of their levels during the transition of the stolon into tuber, and induction of cell expansion upon exogenous jasmonates treatment. However, to date there is only little evidence of the presence of the jasmonic acid-biosynthetic enzymes in stolons or young tubers. As allene oxide cyclase represents the major control point for jasmonic acid biosynthesis, we studied the occurrence of allene oxide cyclase by immunological approaches in the early stages of tuber formation. In developing stolons, allene oxide cyclase as well as lipoxygenase were clearly detectable, but their levels did not change during development. Jasmonic acid treatment for 24h, however, increased lipoxygenase and allene oxide cyclase protein levels in both developmental stages analyzed. In longitudinal sections of stolons of stages 1 and 2, allene oxide cyclase and lipoxygenase occurred in the apex and along the stolon axis. Allene oxide cyclase was clearly detectable in epidermal, cortical and pith parenchymatic cells, showing the highest levels in vascular tissues surrounding cells. Lipoxygenase was mainly located in the parenchymatic cortex cells. The occurrence of allene oxide cyclase in stolons together with the previous identification of jasmonates from developing stolons reveals that these organs are capable to synthesize and metabolize jasmonates.

Loss of At4 function impacts phosphate distribution between the roots and the shoots during phosphate starvation

Plants display a range of adaptive responses to phosphate (Pi) starvation including an increase in the proportion of Pi allocated to the roots, which enhances lateral root development and consequently Pi acquisition. The mechanisms by which plants sense Pi and signal Pi reallocation are largely unknown. Previously, we cloned At4, a gene predicted to contain multiple short open-reading frames (ORFs), whose expression is strongly induced by Pi starvation. At4 is a member of a small gene family whose members, AtIPS1 and two additional genes reported here, At4.1 and At4.2, share little conservation among the predicted ORFs but high conservation of a 22-nt sequence located in the 3' half of the transcript. Here, we show that under Pi-starvation conditions, At4 is expressed in the vascular tissue and transcript levels are regulated by both cytokinin and ABA. at4, an At4 loss-of-function mutant fails to redistribute Pi to the roots correctly in response to Pi deprivation and At4 shoots continue to accumulate a greater proportion of Pi relative to wild type. Consistent with this, the primary root growth rate in at4 is faster than wild type in low-Pi conditions. The conserved sequence found in all members of the At4 gene family hybridizes to a small RNA present in Pi-starved roots. These data support a role for At4 in the internal allocation of Pi and suggest that the At4 gene is not only subject to Pi-starvation-inducible expression, but that transcript levels may be adjusted at a post-transcriptional level by the activity of an miRNA.

PROGRESSIVE EXERCISE PRECONDITIONING PROTECTS AGAINST CIRCULATORY SHOCK DURING EXPERIMENTAL HEATSTROKE

Heat shock protein (HSP) 72 expression protects against arterial hypotension in rat heatstroke. HSP72 can also be induced in multiple organs, including hearts from rats with endurance exercise. We validated the hypothesis that progressive exercise preconditioning may confer cardiovascular protection during heatstroke by inducing the overexpression of HSP72 in multiple organs. To deal with the matter, we assessed the effects of heatstroke on mean arterial pressure, heart rate, cardiac output, stroke volume, total peripheral vascular resistance, colonic temperature, blood gases, and serum or tissue levels of tumor necrosis factor-alpha (TNF-alpha) in urethane-anesthetized rats pretreated without or with progressive exercise training for 1, 2, or 3 weeks. In addition, HSP72 expression in multiple organs was determined in different groups of animals. Heatstroke was induced by exposing the rats to a high blanket temperature (43 degrees C); the moment at which mean arterial pressure decreased from the peak value was taken as the time of heatstroke onset. Previous exercise training for 3 weeks, but not 1 or 2 weeks, conferred significant protection against hyperthermia, arterial hypotension, decreased cardiac output, decreased stroke volume, decreased peripheral vascular resistance, and increased levels of serum or tissue TNF-alpha during heatstroke and correlated with overexpression of HSP72 in multiple organs, including heart, liver, and adrenal gland. However, 10 days after 3 weeks of progressive exercise training, when HSP72 expression in multiple organs returned to basal values, the beneficial effects exerted by 3 weeks of exercise training were no longer observed. These results strongly suggest that HSP72 preconditioning with progressive exercise training protects against hyperthermia, circulatory shock, and TNF-alpha overproduction during heatstroke.

Dysfunction of Endothelial Nitric Oxide Synthase and Atherosclerosis

Atherosclerosis is associated with an impairment of endothelium-dependent relaxations, which represents the reduced bioavailability of nitric oxide (NO) produced from endothelial NO synthase (eNOS). Among various mechanisms implicated in the impaired EDR in atherosclerosis, superoxide generated from dysfunctional eNOS has attracted attention. Under conditions in which vascular tissue levels of tetrahydrobiopterin (BH4), a cofactor for NOS, are deficient or lacking, eNOS becomes dysfunctional and produces superoxide rather than NO. Experimental studies in vitro have revealed that NO from eNOS constitutes an anti-atherogenic molecule. A deficiency of eNOS was demonstrated to accelerate atherosclerotic lesion formation in eNOS knockout mice. In contrast, eNOS overexpression with hypercholesterolemia may promote atherogenesis via increased superoxide generation from dysfunctional eNOS. Thus, eNOS may have 2 faces in the pathophysiology of atherosclerosis depending on tissue BH4 metabolisms. An improved understanding of tissue BH4 metabolisms in atherosclerotic vessels is needed, which would help in developing new strategies for the inhibition and treatment of atherosclerosis.

Malfunction of Vascular Control in Lifestyle-Related Diseases: Endothelial Nitric Oxide (NO) Synthase/NO System in Atherosclerosis

Nitric oxide (NO) from the endothelial NO synthase (eNOS) is believed to be implicated in the development and progression of atherosclerosis. The impaired endothelium-dependent vasodilatory response (EDR) has been demonstrated in vessels exposed to hypercholesterolemia and atherosclerosis. The extent of impairment serves as a predictor of future progression of atherosclerosis. As to the mechanisms of impaired EDR, increased production of superoxide is important. Recently it was revealed that eNOS becomes dysfunctional and produces superoxide rather than NO under conditions in which vascular tissue levels of tetrahydrobiopterin (BH4), a co-factor for eNOS, are deficient or lacking. Dysfunctional eNOS is closely implicated in the endothelial dysfunction represented by impaired EDR in various vascular disorders including atherosclerosis. Regarding the role of eNOS in atherogenesis, experimental studies in vitro have revealed that NO from eNOS constitutes as an anti-atherogenic molecule. In eNOS-knockout mice, eNOS deficiency augments atherosclerotic lesion formation, although the effects may be partly due to the associated hypertension. However, in eNOS-transgenic mice (eNOS-Tg) crossbred with apolipoprotein E-deficient mice (apoE-KO/eNOS-Tg), we found the accelerated lesion formation in association with increased superoxide production from vessels compared with apoE-KO mice. The vascular tissue levels of BH4 were reduced and BH2, an oxidized form, levels were increased. Chronic administration of exogenous BH4 or overexpression of GTPCH-1, a rate limiting enzyme for BH4 synthesis, restored the lesion to the levels comparable to apoE-KO mice. Therefore, eNOS may have two faces in the pathophysiology of atherosclerosis depending on tissue BH4 levels.

Molecular characteristics and differential expression of two nuclear factors containing the FHA domain in Arabidopsis

The forkhead-associated (FHA) domain identified in a wide variety of proteins is a small module that recognizes phosphothreonine epitopes on proteins. AtFHA1 and AtFHA2 of Arabidopsis are homologs of tobacco NtFHA1 encoding a FHA domain-containing transcription activator. Previously, we showed that the FHA domain of NtFHA1 functionally substitutes for that of the Fhl1 forkhead transcription factor involved in rRNA processing in yeast, despite significant differences in their protein structures. In this study, we characterized AtFHA1 and AtFHA2 of Arabidopsis and analyzed their expression patterns. AtFHA1 and AtFHA2 contain an N-terminal FHA domain and a C-terminal acidic region, but lack any known DNA-binding motifs. Both proteins were targeted to the nucleus. GUS staining of transgenic Arabidopsis plants containing the promoter- GUS fusion gene demonstrated high expression of AtFHA2 in whole tissues throughout plant development. In contrast, AtFHA1 was temporally expressed at low levels in roots and vascular tissues of stems. Interestingly, AtFHA1 expression was significantly induced in whole plants in response to external stimuli. The observed differential expression of AtFHA1 and AtFHA2 implies that these two genes have distinct regulatory roles in Arabidopsis.

Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development.

Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf(-/-) mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.

Overexpression of the focal adhesion kinase (p125FAK) in the vascular smooth muscle cells of intimal hyperplasia

Purpose: The migration and proliferation of vascular smooth muscle cells (VSMCs) are important events in the development of intimal hyperplasia (IH). The focal adhesion kinase (FAK) gene encodes a protein tyrosine kinase (p125FAK) involved in signal transduction pathways used in cell adhesion, motility, and proliferation. Because alterations in these cellular processes are thought to occur in VSMCs during IH, we studied FAK expression in healthy arteries and veins in comparison with that in pathologic vessels containing IH. Methods: To determine p125FAK expression at the cellular level, we developed a monoclonal antibody that specifically detected FAK in formalin-fixed, paraffin-embedded tissue sections (5 μm) and analyzed the levels of FAK expression in human arteries and veins. Specificity of monoclonal antibody 4.47 was demonstrated by means of immunofluorescence microscopy showing FAK-specific staining at focal adhesions of healthy human vascular smooth muscle cells (AoSMCs). By using immunohistochemistry techniques, we analyzed the expression of p125FAK in 25 adult human vascular tissue samples from individual patients, which contained a histologically confirmed healthy artery, vein, or IH. Results: FAK expression in healthy and pathologic human vascular tissue was localized predominantly within VSMC cytoplasm. In healthy human artery and vein, borderline FAK expression was detected in the media of seven of 17 vessels and undetectable in the remainder of specimens. However, in vessels containing IH, FAK was overexpressed in the pathologic VSMC populations at moderate-to-strong levels in eight of eight specimens. The levels of FAK expression were directly correlated with structures containing IH, and the results of FAK staining intensity and the percentage of positive cells in these samples were significantly increased compared with normal vascular tissue levels (P <.05, Student t test). Conclusion: These results provide the first evidence that FAK is overexpressed in VSMCs involved in IH and suggest that FAK upregulation may be part of a mechanism for migration and proliferation of VSMCs during this process. Furthermore, the dramatic upregulation of FAK in IH and the relative lack of expression in healthy vessels suggest that FAK may be a rational target for controlling IH. (J Vasc Surg 2001;34:344-9.)

Model of cardiovascular injury in magnesium deficiency

Magnesium deficiency is known to produce cardiovascular lesions. It is, however, not clear as to what constitutes magnesium deficiency – reduced serum levels, reduced tissue levels or reduced intracellular levels of the ionic form of the element. This article cites evidence in support of a hypothesis that a fall in serum magnesium levels may trigger a temporal sequence of events involving vasoconstriction, hemodynamic alterations and vascular endothelial injury to produce pro-inflammatory, pro-oxidant and pro-fibrogenic effects, resulting in initial perivascular myocardial fibrosis which, in turn, would cause myocardial damage and replacement fibrosis. Further, angiotensin II may be the prime mover of the pathogenetic cascade in magnesium deficiency. Importantly, such a mechanism of cardiovascular injury would be independent of a reduction in myocardial or vascular tissue levels of magnesium.

Altered paracrine effect of endothelin in blood vessels of the hyperinsulinemic, insulin resistant obese Zucker rat.

Earlier, we reported that high insulin incubation in vitro leads to increased ETA receptor expression in cultured rat aortic smooth muscle cells (Diabetes 1998, 47: 934-944). Our later observation of enhanced endothelin-1 evoked vasoconstriction in aorta from the hyperinsulinemic obese Zucker rat indicated that this interaction might also be relevant in vivo. To further examine the relationship between insulinemia and endothelin, we characterized endothelin receptor expression and endothelin-1 peptide levels in vascular tissues and plasma from young and old obese Zucker rats. 12 and 40-week-old Zucker obese and lean rats were used. Plasma endothelin-1 levels and endothelin-1 peptide content in the mesenteric artery and in the thoracic aorta were examined by radioimmunoassay. Messenger RNA levels of endothelin-1 peptide and ETA and ETB receptors were examined in the aortic and mesenteric vessels using RT-PCR. Obese rats from both age groups had significantly higher plasma levels of insulin (4-10 fold), total cholesterol (2-3 fold), triglycerides (10-fold), and glucose (approximately 1.5 fold) than their lean counterparts. There was a trend toward worsening lipoproteinemia and glycemia, but improved insulinemia with age in the obese rats. In association with these changes, obese rats exhibited attenuated endothelin-1 peptide and preproET-1 mRNA levels, but conversely elevated ETA and ETB receptor mRNA levels in both aortic and mesenteric vessels. These data suggest that vascular tissue from the metabolically dysregulated obese Zucker rat exhibits attenuated endothelin-1 peptide production and elevated endothelin receptor levels. Since elevated insulin levels have been linked to increased endothelin receptor expression, it is plausible that hyperinsulinemia upregulates endothelin receptors contributing to elevated vasoconstrictor responses to endothelin-1 in this model of obesity and hypertension.

D-α-Tocopherol prevents the hyperglycemia induced activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular smooth muscle cell by an increase of DAG kinase activity

We have reported that d-α-tocopherol can prevent hyperglycemia-induced activation of DAG and PKC levels in vascular tissues as well as normalizing retinal blood flow and renal hyperfiltration. The mechanism of this effect, however, is not clear. Aside from α-tocopherol’s principal role as an antioxidant agent, it has also been shown to act as a membrane stabilizer. Another possibility is that the effect of α-tocopherol is focused on the activation of DAG kinase, which is a key enzyme in the metabolism of DAG. Therefore, in this study, we examined the effect of α-tocopherol on the DAG kinase activity in vascular smooth muscle cell. We have also examined the effect of α-tocopherol, its analogues, and probucol on DAG kinase activities and expression. The present study showed that d-α-tocopherol’s inhibitory effect on DAG-PKC pathway is by increasing DAG kinase activity in rat and human vascular smooth muscle cell (VSMC). Total DAG level was increased by 40±10% (mean±S.E.) ( P<0.05) in human VSMC, after exposure to 22 vs 5 mM glucose. This increase was normalized by d-α-tocopherol treatment in a concentration-dependent manner. In parallel, DAG kinase activation by d-α-tocopherol was also induced in a time- and dose-dependent manner. DAG kinase activity was increased by 57±19% ( P<0.05) in human VSMC and 112±35% ( P<0.05) in rat VSMC after 24 h of incubation with d-α-tocopherol (100 μg/ml). Another lipophilic antioxidant, probucol, also increased DAG kinase activity by 124±34%, but other vitamin E analogues with much less antioxidant potencies were ineffective. Western blots of various DAG kinase isoforms were not changed by d-α-tocopherol treatment. These results provide strong and detailed evidence that d-α-tocopherol can prevent hyperglycemia induced DAG-PKC activation by enhancing DAG kinase activity, probably through an antioxidant effect.

Reduced ischemia–reperfusion injury with rolipram in rat cadaver lung donors: effect of cyclic adenosine monophosphate

Background. The perfusion of rat lungs retrieved from cadavers with a solution containing isoproterenol has been shown to ameliorate the ischemia–reperfusion injury seen in lungs retrieved after death, and this protective effect parallels increases in tissue cyclic adenosine monophosphate levels. In this study, we investigated the effect of rolipram, a phosphodiesterase inhibitor, on capillary permeability and lung cyclic adenosine monophosphate levels in lungs retrieved from circulation-arrested rats. Methods. Using an isolated perfused lung circuit, we retrieved lungs from circulation-arrested donor rats either ventilated with 100% oxygen or not ventilated for varying postmortem times. The lungs were reperfused with or without rolipram (2 μmol/L). The capillary filtration coefficient and wet to dry weight ratio, indicators of pulmonary vascular integrity, were determined, and tissue levels of adenine nucleotides and cyclic adenosine monophosphate were measured by high-performance liquid chromatography. Results. The capillary filtration coefficient was significantly reduced in nonventilated cadaver lungs reperfused with rolipram 120 minutes after death ( p < 0.05). Oxygen ventilation or reperfusion with rolipram had a similar effect on the capillary filtration coefficient. Cyclic adenosine monophosphate levels were significantly higher in rolipram-reperfused lungs retrieved 120 minutes after death in both oxygen-ventilated ( p < 0.01) and nonventilated ( p < 0.01) lungs. Conclusions. In lungs from nonventilated, circulation-arrested donors, reperfusion with rolipram reduces the ischemia–reperfusion injury that may be due to intracellular cyclic adenosine monophosphate. Alteration of perfusate may have an impact on capillary leak caused by antecedent ischemia. Thus, rolipram may be a useful adjunct in the preservation of donor lungs retrieved after death.

Reduction-oxidation (Redox) and vascular tissue level of homocyst(e)ine in human coronary atherosclerotic lesions and role in extracellular matrix remodeling and vascular tone.

Hyperhomocyst(e)inemia in patients with coronary and peripheral arterial occlusion has been demonstrated by others. Redox-state of homocyst(e)ine causes dysfunction of endothelial cells and promote growth of vascular smooth muscle cells. The role of tissue, protein bound and unbound, oxidative mixed disulfides in the development of fibrous plaque in atherosclerotic lesion is not known. Redox-state around the fibroblasts and vascular smooth muscle cells modulates the expression of extracellular matrix (ECM) components (Tyagi et al. 1996, J Cell Biochem, 61: 139-151). To determine the role of tissue homocystine in fibrotic atherosclerotic plaque development, coronary arteries were isolated from ischemic explanted hearts (n = 10). Apparently normal vascular tissue was obtained from idiopathic cardiomyopathic explanted hearts (n = 10). Tissue extract were prepared from atherosclerotic lesions and from normal arteries devoid of adventitia. Interaction of homocystine with Ellman's reagent (5, 5'-dithio-bis-2-nitro benzoic acid) catalyzed by limiting amount of reducing agent (catalyst) generated change in optical density (OD) at 412 nm in dose dependent fashion. We have generated a standard curve between change at 412 nm and amount of homocystine. The change in OD at 412 nm with increasing amount (0-25 microg) of homocystine demonstrated linearity. The protein-bound oxidized disulfides were precipitated by trichloroacetic acid (TCA) and free-oxidative disulfides in the supernatant were collected. The pathophysiological amount of protein-bound disulfide in atherosclerotic tissue (1.0 +/- 0.2 microg/mg total protein) was 10 times that in normal tissue (0.1 +/- 0.01 microg/mg, p < 0.001). The amount of free oxidative disulfide in atherosclerotic tissue (1.5 +/- 0.3 microg/mg) was 15 times that in normal tissue (0.12 +/- 0.02 microg/mg, p < 0.001). To determine the role of homocystine in ECM expression, ECM collagenase activity in the presence and absence of homocystine was measured by zymography. The effect of homocysteine on collagenase activity was biphasic, increased at < [0.01 mM] and inhibited at > [0.1 mM]. To determine whether homocystine regulates vascular tone, isometric measurements were carried out using normal coronary rings. Results suggested that homocystine induced endothelial-modulated vasoconstriction in coronary vessels. Tissue oxidative disulfides and the homocystine may contribute to the development of fibrotic atherosclerotic lesions and vascular dysfunction.