Dietary selenium increases the antioxidant levels and ATPase activity in the arteries and veins of poultry.

Selenium (Se) deficiency is associated with the pathogenesis of vascular diseases. It has been shown that oxidative levels and ATPase activity were involved in Se deficiency diseases in humans and mammals; however, the mechanism by how Se influences the oxidative levels and ATPase activity in the poultry vasculature is unclear. We assessed the effects of dietary Se deficiency on the oxidative stress parameters (superoxide dismutase, catalase, and hydroxyl radical) and ATPase (Na(+)K(+)-ATPase, Ca(++)-ATPase, Mg(++)-ATPase, and Ca(++)Mg(++)-ATPase) activity in broiler poultry. A total of 40 broilers (1-day old) were randomly divided into a Se-deficient group (L group, fed a Se-deficient diet containing 0.08 mg/kg Se) and a control group (C group, fed a diet containing sodium selenite at 0.20 mg/kg Se). Then, arteries and veins were collected following euthanasia when typical symptoms of Se deficiency appeared. Antioxidant indexes and ATPase activity were evaluated using standard assays in arteries and veins. The results indicated that superoxide dismutase activity in the artery according to dietary Se deficiency was significantly lower (p < 0.05) compared with the C group. The catalase activity in the veins and hydroxyl radical inhibition in the arteries and veins by dietary Se deficiency were significantly higher (p < 0.05) compared with the C group. The Se-deficient group showed a significantly lower (p < 0.05) tendency in Na(+)K(+)-ATPase activity, Ca(++)-ATPase activity, and Ca(++)Mg(++)-ATPase activity. There were strong correlations between antioxidant indexes and Ca(++)-ATPase activity. Thus, these results indicate that antioxidant indexes and ATPases may have special roles in broiler artery and vein injuries under Se deficiency.

Somatic Activating PIK3CA Mutations Cause Venous Malformation.

Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects.

Matrix Metalloproteinase 9 (MMP-9) Regulates Vein Wall Biomechanics in Murine Thrombus Resolution.

OBJECTIVE: Deep venous thrombosis is a common vascular problem with long-term complications including post-thrombotic syndrome. Post-thrombotic syndrome consists of leg pain, swelling and ulceration that is related to incomplete or maladaptive resolution of the venous thrombus as well as loss of compliance of the vein wall. We examine the role of metalloproteinase-9 (MMP-9), a gene important in extracellular remodeling in other vascular diseases, in mediating thrombus resolution and biomechanical changes of the vein wall. METHODS AND RESULTS: The effects of targeted deletion of MMP-9 were studied in an in vivo murine model of thrombus resolution using the FVB strain of mice. MMP-9 expression and activity significantly increased on day 3 after DVT. The lack of MMP-9 impaired thrombus resolution by 27% and this phenotype was rescued by the transplantation of wildtype bone marrow cells. Using novel biomechanical techniques, we demonstrated that the lack of MMP-9 significantly decreased thrombus-induced loss of vein wall compliance. Biomechanical analysis of the contribution of individual structural components showed that MMP-9 affected the elasticity of the extracellular matrix and collagen-elastin fibers. Biochemical and histological analyses correlated with these biomechanical effects as thrombi of mice lacking MMP-9 had significantly fewer macrophages and collagen as compared to those of wildtype mice. CONCLUSIONS: MMP-9 mediates thrombus-induced loss of vein wall compliance by increasing stiffness of the extracellular matrix and collagen-elastin fibers during thrombus resolution. MMP-9 also mediates macrophage and collagen content of the resolving thrombus and bone-marrow derived MMP-9 plays a role in resolution of thrombus mass. These disparate effects of MMP-9 on various aspects of thrombus illustrate the complexity of individual protease function on biomechanical and morphometric aspects of thrombus resolution.

Elevated guanylate cyclase and cyclic-guanosine monophosphate-dependent protein kinase levels in nasal mucosae of antigen-challenged rats.

OBJECTIVE: In patients with severe allergic rhinitis, the most serious symptom is rhinostenosis, which is considered to be induced by a dilatation of plexus cavernosum. The vascular relaxing responses to chemical mediators are mainly mediated by the production of nitric oxide (NO). However, the exact mechanism(s) in nasal venoresponsiveness of allergic rhinitis is not fully understood. In the present study, we investigated the roles of soluble guanylate cyclase (sGC) and cyclic-guanosine monophosphate (c-GMP)-dependent protein kinase G (PKG) in venodilatation of nasal mucosae of antigen-challenged rats. METHODS: Actively sensitized rats were repeatedly challenged with aerosolized antigen (2,4-dinitrophenylated Ascaris suum). Twenty-four hours after the final antigen challenge, nasal septum mucosa was exposed surgically and observed directly in vivo under a stereoscopic microscope. The sodium nitroprusside (SNP) and 8-Br-cGMP (a PKG activator) were administered into arterial injection, and the venous diameters of nasal mucosa were observed. RESULTS: The intra-arterial injections of SNP and 8-Br-cGMP-induced venodilatation were significantly augmented in the nasal mucosae of repeatedly antigen-challenged rats. Furthermore, protein expressions of sGC and PKG were significantly increased in nasal mucosae of the antigen-challenged rats. CONCLUSION: The present findings suggest the idea that the promoted cGMP/PKG pathway may be involved in the enhanced NO-induced venodilatation in nasal mucosae of antigen-challenged rats.

Matrix metalloproteinase-9 deletion is associated with decreased mid-term vein wall fibrosis in experimental stasis DVT.

INTRODUCTION: Post thrombotic syndrome therapy is primarily palliative, and the associated vein wall inflammatory mechanisms are unclear. Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP9 directly contributes to vein wall remodeling after VT is unknown. METHODS: WT and MMP9 -/- mice underwent stasis VT by ligation of the inferior vena cava (IVC) and tissue was harvested at 2, 8, and 21days. Assessment of thrombus size, and gene, protein and structural vein wall determinations were done. RESULTS: VT resolution was increased in MMP9-/- mice as compared with controls at 21d only. The primary phenotypic fibrotic vein wall differences occurred at 8d post VT, with significantly less vein wall collagen content as assessed by Picosirius red staining in MMP9 -/- mice as compared with WT. Increased monocytic vein wall influx with less IL-1b and TGFb was found in MMP9 -/- vein walls as compared with WT. Corresponding levels of PAI-1 were increased in MMP9 -/- compared with WT, and no difference in FSP-1+cells as compared with controls. CONCLUSIONS: In stasis VT, MMP9 modulates midterm vein wall collagen content, with an altered local inflammatory and profibrotic environment, likely directed by monocytes. Thus, MMP9 plays a role in both vein wall responses as well as late thrombus resolution.

Cyclic adenosine monophosphate response-element binding protein activation by mitogen-activated protein kinase-activated protein kinase 3 and four-and-a-half LIM domains 5 plays a key role for vein graft intimal hyperplasia.

OBJECTIVE: Intimal hyperplasia (IH) is the main cause of vein graft stenosis or failure after bypass surgery. Basic investigations are proceeding in an animal model of mechanically desquamated arteries, and numerous molecules for potential IH treatments have been identified; however, neither insights into the mechanism of IH nor substantially effective treatments for its suppression have been developed. The goals of the present study are to use human vein graft samples to identify therapeutic target genes that control IH and to investigate the therapeutic efficacy of these candidate molecules in animal models. METHODS: Using microarray analysis of human vein graft samples, we identified two previously unrecognized IH-related genes, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) and four-and-a-half LIM domains 5 (FHL5). RESULTS: Transfer of either candidate gene resulted in significantly elevated vascular smooth muscle cell (VSMC) proliferation and migration. Interestingly, cotransfection of both genes increased VSMC proliferation in an additive manner. These genes activated cyclic adenosine monophosphate response-element (CRE) binding protein (CREB), but their mechanisms of activation were different. MAPKAPK3 phosphorylated CREB, but FHL5 bound directly to CREB. A CREB dominant-negative protein, KCREB, which blocks its ability to bind CRE, repressed VSMC proliferation and migration. In a wire-injury mouse model, gene transfer of KCREB plasmid significantly repressed IH. In this vessel tissue, CRE-activated gene expression was repressed. Furthermore, we confirmed the changes in MAPKAPK3 and FHL5 expression using vein graft samples from eight patients. CONCLUSIONS: We successively identified two previously unrecognized IH activators, MAPKAPK3 and FHL5, using human vein graft samples. Gene transfer of KCREB repressed IH in an animal model. Inhibition of CREB function is a promising gene therapy strategy for IH.

Bioassay-comparison of the antioxidant efficacy of hydrogen sulfide and superoxide dismutase in isolated arteries and veins.

Recent studies suggest that hydrogen sulfide (H2S) exhibits potent antioxidant capacity and improves vascular and tissue functions. Thus we aimed to compare the antioxidant efficacy of H2S to that of superoxide dismutase (SOD).Isometric force of isolated rat carotid arteries and gracilis veins was measured with a myograph. The vasomotor effect of the superoxide-generator pyrogallol (10-5M) was obtained in control conditions, and then in the presence of SOD (120 U/ml) or H2S (10-5M or 10-4M), respectively. Spectrophotometric measurements were performed to detect the effect of SOD and H2S on the auto-oxidation of pyrogallol.Pyrogallol increased the isometric force of carotid arteries (9.7 ± 0.8 mN), which was abolished by SOD (5.3 ± 0.8 mN), was not affected by 10-5M H2S (9.1 ± 0.5 mN), whereas 10-4M H2S slightly, but significantly reduced it (8.1 ± 0.7 mN). Pyrogallol significantly increased the isometric force of gracilis veins (1.3 ± 0.2 mN), which was abolished by SOD (0.9 ± 0.2 mN), whereas 10-5M (1.3 ± 0.2 mN), or 10-4M H2S (1.2 ± 0.2 mN) did not affect it. Pyrogallol-induced superoxide production was measured by a spectrophotometer (A420 = 0.19 ± 0.0). SOD reduced absorbance (A420 = 0.02 ± 0.0), whereas 10-5M H2S did not (A420 = 0.18 ± 0.0) and 10-4M H2S slightly reduced it (A420 = 0.15 ± 0.0).These data suggest that H2S is a less effective vascular antioxidant than SOD. We propose that the previously described beneficial effects of H2S are unlikely to be related to its direct effect on superoxide.

Expression of NOS isoforms in internal spermatic veins of infertile men with varicocele.

Although varicocele is a relatively common entity encountered in the evaluation of infertile men, the exact pathophysiology still remains unclear. Recently, as previously widely investigated in various parts of human circulatory system, nitric oxide synthase (NOS) and its product, nitric oxide (NO) have been thought to play a role in the development of varicocele and thus male infertility. In this study, we determined the concentration of NO metabolite and the expression of NOS isoforms in the internal spermatic (ISV) and superficial branch of inferior epigastric veins of infertile men with varicocele. The study included 60 infertile men with clinically unilateral or bilateral varicocele. Expression of inducible and endothelial NOS (iNOS and eNOS) isoforms were investigated in tissue arrays of internal spermatic and superficial branch of inferior epigastric veins with immunohistochemistry. NO metabolite (nitrite) levels were measured using the calorimetric method. A significantly higher expression of eNOS was observed in the varicose veins (mean score: 2.25 and 1.55, respectively; p = 0.0001). However, statistically, there was no significant difference for expression of iNOS between varicose and control veins (p = 0.094). The nitrite concentration and NOS expression were not found to be correlated with clinical variables (varicocele grade, maximum varicose vein diameter, and sperm concentration, motility, and morphology) (p > 0.05). As a result, the significantly higher expression of eNOS in ISV may be responsible for the development of varicocele, although this finding is not accompanied by an increase in NO concentration. Still, the pattern of the relationship between varicocele and increased eNOS expression warrants further investigation.

Elevation of both reactive oxygen species and antioxidant enzymes in vein tissue of infertile men with varicocele.

OBJECTIVE: To compare reactive oxygen species (ROS) and antioxidants in internal spermatic vein tissue of men with varicocele and a control group with inguinal hernia. Also to compare the levels of oxidants and antioxidants in infertile and fertile men with varicocele. PATIENTS AND METHODS: 48 varicocele patients and 12 non-varicocele inguinal hernia patients participated in this study. The varicocele group was again divided into two groups--fertile men with varicocele and infertile men with varicocele. Internal spermatic vein tissue samples were obtained. The level of the malondialdehyde (MDA), an indicator of oxidative stress, and the activities of antioxidant enzymes (defense systems) against oxidative stress such as superoxide dismutase (SOD) and catalase (CAT) were estimated in these tissue samples. RESULTS: The mean level of MDA in the varicocele group was significantly higher than in the hernia group, whereas the levels of antioxidants (SOD and CAT) were significantly lower in the varicocele group than in the hernia group. When compared with the subgroups of the varicocele group, the MDA levels and SOD and CAT activities were significantly higher in the infertile varicocele group than in the fertile varicocele group. CONCLUSIONS: Antioxidant enzyme levels generally decrease due to a high consumption in varicocele patients. On the contrary, in our study both MDA and antioxidant enzymes increased in the internal spermatic venous wall of infertile varicocele patients. This situation may be explained by an adaptation against oxidative stress in varicocele, which could be defined as a chronic process.

The mechanical stress-activated serum-, glucocorticoid-regulated kinase 1 contributes to neointima formation in vein grafts.

RATIONALE: Mechanical stress plays an important role in proliferation of venous smooth muscle cells (SMCs) in neointima, a process of formation that contributes to failure of vein grafts. However, it is unknown what intracellular growth signal leads to proliferation of venous SMCs. OBJECTIVE: The objective of this study is to identify mechanisms of mechanical stretch on neointima formation. METHODS AND RESULTS: By a microarray analysis, we found that mechanical cyclic stretch (15% elongation) stimulated the transcription of SGK-1 (serum-, glucocorticoid-regulated kinase-1). Mechanical stretch-induced SGK-1 mRNA expression was blocked by actinomycin D. The mechanism for the SGK-1 expression involved MEK1 but not p38 or JNK signaling pathway. SGK-1 activation in response to stretch is blocked by insulin-like growth factor (IGF)-1 receptor inhibitor and mammalian target of rapamycin complex (mTORC)2 inhibitor (Ku-0063794) but not mTORC1 inhibitor (rapamycin). Mechanical stretch-induced bromodeoxyuridine incorporation was reduced by 83.5% in venous SMCs isolated from SGK-1 knockout mice. In contrast, inhibition of Akt, another downstream signal of PI3K resulted in only partial inhibition of mechanical stretch-induced proliferation of venous SMCs. Mechanical stretch also induced phosphorylation and nuclear exportation of p27(kip1), whereas knockout of SGK-1 attenuated this effect of mechanical stretch on p27(kip1). In vivo, we found that placement of a vein graft into artery increased SGK-1 expression. Knockout of SGK-1 effectively prevented neointima formation in vein graft. There is significant lower level of p27(kip1) located in the nucleus of neointima cells in SGK-1 knockout mice compared with that of wild-type vein graft. In addition, we also found that wire injury of artery or growth factors in vitro increased expression of SGK-1. CONCLUSIONS: These results suggest that SGK-1 is an injury-responsive kinase that could mediate mechanical stretch-induced proliferation of vascular cells in vein graft, leading to neointima formation.

Vein tissue expression of matrix metalloproteinase as biomarker for hemodialysis arteriovenous fistula maturation.

Failure of arteriovenous fistula (AVF) maturation is attributed to impaired vein remodeling. The purpose of this study is to identify whether vein matrix metalloproteinase (MMP) expression and activity is associated with AVF maturation. Patients with renal insufficiency undergoing surgery had their vein segments harvested and snap-frozen at time of AVF construction. Expression of MMP-2, MMP-9, membrane type-1 MMP (MT1-MMP), tissue inhibitor of metallopreoteinases type 2 (TIMP-2), and TIMP-4 were measured using zymography and Western blotting techniques. Of 14 patients enrolled, 9 had successful maturation and 5 had failure of AVF maturation. Significantly higher levels of MT1-MMP (an MMP-2 activator; P = .01), TIMP-2 (an MMP-2 inhibitor; P = .03), MMP-2 latent (P = .02), and MMP-2 total (P = .03) were associated with AVF maturation. There was a trend toward higher levels of TIMP-4 in the successful group (P = .18). These data demonstrate a positive relationship between MMP-2 expression in veins and AVF maturation. MMP-2 could serve as a potential preoperative marker to predict maturation.

Rho kinase activation and ROS production contributes to the cooling enhanced contraction in cutaneous equine digital veins.

A decrease in environmental temperature can directly affect the contractility of cutaneous vasculature, mediated in part by alpha(2)-adrenoceptors. Most of the cellular mechanisms underlying the cooling-enhanced contractility to alpha(2)-adrenoceptor agonists have been reported in cutaneous arteries but little information is available on cutaneous veins. To investigate the cellular mechanisms associated with the cooling-enhanced contraction to UK-14304 (alpha(2)-adrenoceptor agonist), isolated equine digital veins (EDVs) were studied at 30 degrees C and 22 degrees C. The effects of inhibitors were studied on the contractile response to UK-14304 (0.1 microM). The cooling-enhanced responses were inhibited by Rho kinase inhibitors [maximum response to UK-14304 95.2 +/- 8% of response to depolarizing Krebs solution (DKS) in control vessels cooled to 22 degrees C, compared with 31.4 +/- 6% in the presence of fasudil 1 microM and 75.8 +/- 6% with Y-27632 0.1 microM] and the effects of these inhibitors were considerably less at 30 degrees C (control response 56.4 +/- 5% of DKS; 34.9 +/- 6% with fasudil 1 microM and 50.6 +/- 9% with Y-27632 0.1 microM). Furthermore, Western blotting showed that one of the downstream targets for Rho kinase activity, ezrin/radixin/moesin, was phosphorylated after cooling and reduced by fasudil (1 microM) only at 22 degrees C. The activation of protein kinase C contributed to the contractile response, but predominantly at 30 degrees C (maximum response 82.3 +/- 9% of DKS for control; 57.7 +/- 10% in the presence of chelerythrine 10 microM) with no significant effect at 22 degrees C. The reduction of the response at 22 degrees C by antioxidants, rotenone (14% reduction), and tempol (21% reduction) suggested the contribution of reactive oxygen species (ROS). No evidence was obtained to support the participation of tyrosine kinase. These data demonstrate that Rho kinase activation and the production of ROS contributes to the cooling-enhanced contraction in these cutaneous digital veins.

Endothelial microsomal prostaglandin E synthase-1 exacerbates neuronal loss induced by kainate.

Prostaglandin E(2) (PGE(2)) is increased in the brain after kainic acid (KA) treatment. We previously demonstrated that KA also induces PG synthase cyclooxygenase-2 (COX-2) expression rapidly in neurons of the brain and slowly in astrocytes and endothelia. Prevention of KA-induced neuronal damage by nonneuronal COX-2 inhibition suggests a novel modulatory mechanism for neuronal injury by nonneuronal PGs. It remains unclear, however, which PG synthase is responsible for this modulation following COX-2 synthesis after neuronal insult. In addition, the PG receptor subtype that is involved in neuronal loss remains controversial. Here we demonstrate that microinjection of KA induces microsomal prostaglandin E synthase-1 (mPGES-1) in venous endothelial cells but not in neurons or astrocytes. We found that mPGES-1 plays a central role in delayed production of PGE(2) and that mPGES-1-deficient mice exhibit significantly less neuronal loss induced by KA. Furthermore, KA injection caused an increase in the immunoreactivity for the EP3 receptor in the astrocytic endfeet that surround vascular endothelia. Neurons form intimate interactions with astrocytes via glutamate, and astrocytes contact vascular endothelia through endfeet. These findings suggest that endothelial cells may control neuronal excitotoxicity, most likely by regulating astrocytes via inducible PGE(2).

Arterial-venous specification during development.

The major arteries and veins of the vertebrate circulatory system are formed early in embryonic development, before the onset of circulation, following de novo aggregation of "angioblast" progenitors in a process called vasculogenesis. Initial embryonic determination of artery or vein identity is regulated by variety of genetic factors that work in concert to specify endothelial cell fate, giving rise to 2 distinct components of the circulatory loop possessing unique structural characteristics. Work in multiple in vivo animal model systems has led to a detailed examination of the interacting partners that determine arterial and venous specification. We discuss the hierarchical arrangement of many signaling molecules, including Hedgehog (Hh), vascular endothelial growth factor (VEGF), Notch, and chicken ovalbumin upstream-transcription factor II (COUP-TFII) that promote or inhibit divergent pathways of endothelial cell fate. Elucidation of the functional role of these genetic determinants of blood vessel specification together with the epigenetic factors involved in subsequent modification of arterial-venous identity will allow for potential new therapeutic targets for vascular disorders.

Role of crosstalk between phosphatidylinositol 3-kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways in artery-vein specification.

Functional and structural differences between arteries and veins lie at the core of the circulatory system, both in health and disease. Therefore, understanding how artery and vein cell identities are established is a fundamental biological challenge with significant clinical implications. Molecular genetic studies in zebrafish and other vertebrates in the past decade have begun to reveal in detail the complex network of molecular pathways that specify artery and vein cell fates during embryonic development. Recently, a chemical genetic approach has revealed evidence that artery-vein specification is governed by cross talk between phosphoinositide 3-kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in artery-vein specification. We discuss recent findings on the signaling pathways involved in artery-vein specification during zebrafish development and compare and contrast these results to those from mammalian systems. It is anticipated that the complementary approaches of genetics and chemical biology, involving a variety of model organisms and systems, will lead to a better understanding of artery-vein specification and possibly to novel therapeutic approaches to treat vascular diseases.

Matrix metalloproteinases in venous tissue remodeling and varicose vein formation.

Matrix metalloproteinases (MMPs) play a major role in extracellular matrix (ECM) turnover under both physiological and pathological conditions. Studies on venous tissues from experimental animals and humans identified several MMP subtypes, and showed significant changes in the expression and activity of specific MMPs during vein wall remodeling. Also, significant research has focused on the role of MMPs in chronic venous disease (CVD) and varicose vein formation in the lower extremities and their progression to thrombophlebitis and venous leg ulcer. Several hypotheses have been forwarded regarding the pathophysiological mechanisms underlying the relation between MMPs and the formation, progression and complications of varicose veins. The effects of MMPs on ECM degradation could result in significant venous tissue remodeling and degenerative and structural changes in the vein wall, leading to venous dilation and valve dysfunction. MMPs may also induce early changes in the endothelium and venous smooth muscle function in the absence of significant ECM degradation or structural changes in the vein wall. In addition, evidence suggests increased activity of MMPs in the advanced stages of chronic venous insufficiency (CVI) associated with skin changes and leg ulceration as well as in the wound fluid environment. Several pharmacological therapies and surgical strategies are being utilized in the management of varicose veins, with variable success and recurrence rates. Inhibition of MMPs may represent a novel therapeutic intervention to limit the progression of varicose veins to CVI and leg ulceration.

Mesenchymal stem cells differentiate into an endothelial phenotype, reduce neointimal formation, and enhance endothelial function in a rat vein grafting model.

Autologous vein grafts is still commonly used for arterial reconstructive procedures. Their success is limited by the development of neointimal hyperplasia. Clinical and experimental evidence suggest that the bone marrow derived mesenchymal stem cells (MSCs) participate in the neovascularization. The current study used a direct approach to test the hypothesis that, after vein grafting in a rat model, MSCs have potential effects on reendothelialization and neointimal formation. MSCs were isolated by bone marrow cell adherence. Autologously interpositioning left external jugular vein (LEJV) to left common carotid artery-induced vein grafting model of r at w as utilized. Vascular lesion formation after transplantation of MSCs labeled with 4',6-diamidino-2-phenylindole (DAPI) was investigated. Two weeks after implantation, immunofluorescence studies revealed that engrafted cells acquired an endothelial phenotype, and some expressed endothelial nitric oxide synthase (eNOS). Furthermore, proliferation of cells and neointimal formation decreased significantly after MSC implantation. Real-time reverse transcription-PCR and western blotting analysis showed a rise of eNOS expression in the MSC group compared with the vein grafting group. Therefore, engrafted MSCs appeared to differentiate into endothelial cells, diminish the neointima formation and contribute to the improvement on endothelial function, which indicates that MSCs may exert an important function as repair mechanism in vascular injury after vein grafting.

Inhibition of aldehyde dehydrogenase type 2 attenuates vasodilatory action of nitroglycerin in human veins.

Recent studies suggest that the mitochondrial aldehyde dehydrogenase (ALDH)2 is involved in vascular bioactivation of nitroglycerin (GTN). However, neither expression of ALDH2 nor its functional role in GTN bioactivation has been reported for the main drug target in humans, namely capacitance vessels. We investigated whether ALDH2 is expressed in human veins and whether inhibition of the enzyme attenuates nitroglycerin effects in these vessels. We determined expression of ALDH2 and dehydrogenase activity in human veins by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescence microscopy. In vitro contraction experiments were performed in the presence or absence of the ALDH inhibitors chloral hydrate, cyanamide, and ethoxycyclopropanol. Concentration response curves were determined for the alpha-agonist phenylephrine, nitroglycerin, and the direct NO donor diethylamine NONOate (DEA-NONOate). ALDH2 expression was largely confined to smooth muscle cells as determined by confocal immunofluorescence microscopy. Contractile responses to phenylephrine were unaffected by all ALDH inhibitors tested. In clear contrast, the ALDH inhibitors significantly reduced the potency of nitroglycerin by approximately 1 order of magnitude (P < or = 0.01). Neither of the inhibitors affected the potency of the direct NO donor DEA-NONOate, which ruled out nonspecific effects on the NO signaling cascade. In human capacitance vessels, ALDH2 is a key enzyme in the biotransformation of the frequently used antianginal drug nitroglycerin.

Nitric oxide in flow-through venous flaps and effects of L-arginine and nitro-L-arginine methyl ester (L-NAME) on nitric oxide and flap survival in rabbits.

OBJECT: Venous flaps are relatively recent practices in plastic surgery, and their life mechanisms are not known exactly. Partial necroses frequently occur in these flaps; therefore, their survival should be enhanced. Nitric oxide (NO) is an endogenous compound which has recently been dwelt upon frequently in flap pathophysiology, and its effect on viability in conventional flaps has been demonstrated. However, its role in venous flaps is unknown. The purpose of this study is to determine possible changes in the NO level in venous flaps and to investigate the possible effects of NO synthesis precursor and inhibitor on the venous flap NO level and flap survival. MATERIAL AND METHODS: Thirty white male rabbits of New Zealand type, aged 6 months, were divided into 3 groups as control (n = 10), L-arginine (n = 10), and nitro-L-arginine methyl ester (L-NAME) (n = 10). Blood and tissue samples were taken from one ear of 10 rabbits in the control group for the determination of NO basal levels 2 weeks before flap practice. The 3-x-5-cm flow-through venous flaps, which are sitting on the anterior branch of the central vein, were elevated on each ear of 10 rabbits in all groups. After flaps were sutured to their beds, 2 mL/d saline, 1 g/kg/d L-arginine (NO synthesis precursor), and 50 mg/kg/d L-NAME (NO synthesis inhibitor) were administered intraperitoneally in control, L-arginine, and L-NAME groups, respectively, for 3 days. At the 24th postoperative hour, blood and tissue samples were taken from all animals for biochemical analyses. At day 7, flap survivals were assessed. RESULTS: Mean NO levels in the blood following the flap elevation (129 +/- 76 micromol/mg protein) increased in comparison with basal levels (59 +/- 44 micromol/mg protein) (P < 0.06); however, the tissue level remained unchanged. NO levels in the blood in the L-arginine and L-NAME groups were alike compared with the control group. The tissue NO level in L-NAME group (0.08 +/- 0.03 micromol/mg protein) decreased significantly compared to the control group (0.46 +/- 0.36 micromol/mg protein) (P < 0.001). Mean flap survival in the L-arginine group (95% +/- 6) increased according to the control group (61% +/- 14) (P < 0.001), whereas it did not change in the L-NAME group (55% +/- 13). CONCLUSION: In our model of venous flap, NO level in the blood increased, while it did not change in the tissue; L-arginine significantly enhanced flap viability without affecting NO level. Additionally, L-NAME decreased NO level, but it did not affect flap survival. In light of these findings, NO increases in venous flaps; the change in its level does not affect flap survival, though. However, L-arginine enhances venous flap survival if not by virtue of NO.