The relationship between oxidised LDL, endothelial progenitor cells and coronary endothelial function in patients with CHD.

OBJECTIVE: The balance between coronary endothelial dysfunction and repair is influenced by many protective and deleterious factors circulating in the blood. We studied the relationship between oxidised low-density lipoprotein (oxLDL), circulating endothelial progenitor cells (EPCs) and coronary endothelial function in patients with stable coronary heart disease (CHD). METHODS: 33 patients with stable CHD were studied. Plasma oxLDL was measured using ELISA, coronary endothelial function was assessed using intracoronary acetylcholine infusion and EPCs were quantified using flow cytometry for CD34(+)/KDR(+) cells. RESULTS: Plasma oxLDL correlated positively with the number of EPCs in the blood (r=0.46, p=0.02). There was a positive correlation between the number of circulating EPCs and coronary endothelial function (r=0.42, p=0.04). There was no significant correlation between oxLDL and coronary endothelial function. CONCLUSIONS: Plasma levels of oxLDL are associated with increased circulating EPCs in the blood of patients with CHD, which may reflect a host-repair response to endothelial injury. Patients with stable CHD had a high prevalence of coronary endothelial dysfunction, which was associated with lower numbers of circulating EPCs, suggesting a mechanistic link between endothelial dysfunction and the pathogenesis of atherosclerosis.

Adventitial ablation technique that permits the assessment of adventitial-dependent contribution to microvascular contractile function.

Resistance arteries have been implicated as a major contributing factor in the sequela of disease conditions such as hypertension and diabetes and, as such, are a major focus of cardiovascular research. The paracrine influence of the intimal endothelial layer of resistance arteries is well established. Considering the growing body of evidence substantiating a functionally relevant vascular adventitia, in this study we have established a technique that permits determination of the functional influence of the adventitial layer on resistance artery tone. Isolating adventitial-dependent function, analogous to isolating endothelial function, has potentially significant implications for studying the as yet unexplored role of the microvascular adventitial layer in modulating acute vascular contractile function.

Perivascular mast cells regulate vein graft neointimal formation and remodeling.

Objective. Emerging evidence suggests an important role for mast cells in vein graft failure. This study addressed the hypothesis that perivascular mast cells regulate in situ vascular inflammatory and proliferative responses and subsequent vein graft neointimal lesion formation, using an optimized local mast cell reconstitution method. Methods and Results. Neointimal hyperplasia was induced by insertion of a vein graft into the right carotid artery in wild type and mast cell deficient Kit(W-sh/W-sh) mice. In some experiments, mast cells were reconstituted systemically (tail vein injection of bone marrow-derived mast cells) or locally (directly into the right neck area) prior to vein grafting. Vein graft neointimal lesion formation was significantly (P < 0.05) reduced in Kit(W-sh/W-sh) mice. Mast cell deficiency reduced the number of proliferating cells, and inhibited L-selectin, CCL2, M-CSF and MIP-3α expression in the vein grafts. Local but not systemic mast cell reconstitution restored a perivascular mast cell population that subsequently promoted neointimal formation in mast cell deficient mice. Conclusion. Our data demonstrate that perivascular mast cells play a key role in promoting neointima formation by inducing local acute inflammatory and proliferative responses. These results suggest that ex vivo intraoperative targeting of mast cells may have therapeutic potential for the prevention of pathological vein graft remodeling.

Mast cells and vascular diseases.

Mast cells are increasingly being recognized as effector cells in many cardiovascular conditions. Many mast-cell-derived products such as tryptase and chymase can, through their enzymic action, have detrimental effects on blood vessel structure while mast cell-derived mediators such as cytokines and chemokines can perpetuate vascular inflammation. Mice lacking mast cells have been developed and these are providing an insight into how mast cells are involved in cardiovascular diseases and, as knowledge increase, mast cells may become a viable therapeutic target to slow progression of cardiovascular disease.

Succinobucol-eluting stents increase neointimal thickening and peri-strut inflammation in a porcine coronary model.

OBJECTIVE: The aim of this study was to assess the efficacy of stent-based delivery of succinobucol alone and in combination with rapamycin in a porcine coronary model. BACKGROUND: Current drugs and polymers used to coat coronary stents remain suboptimal in terms of long term efficacy and safety. Succinobucol is a novel derivative of probucol with improved antioxidant and anti-inflammatory properties. METHODS: Polymer-free Yukon stents were coated with 1% succinobucol (SucES), 2% rapamycin (RES), or 1% succinobucol plus 2% rapamycin solutions (SucRES) and compared with a bare metal stent (BMS). RESULTS: The in vivo release profile of SucES indicated drug release up to 28 days (60% drug released at 7 days); 41 stents (BMS, n = 11; SucES, n =10; RES, n = 10; SucRES, n = 10) were implanted in the coronary arteries of 17 pigs. After 28 days, mean neointimal thickness was 0.31 ± 0.14 mm for BMS, 0.51 ± 0.14 mm for SucES, 0.19 ± 0.11 mm for RES, and 0.36 ± 0.17 mm for SucRES (P < 0.05 for SucES vs. BMS). SucES increased inflammation and fibrin deposition compared with BMS (P < 0.05), whereas RES reduced inflammation compared with BMS (P < 0.05). CONCLUSION: In this model, stent-based delivery of 1% succinobucol using a polymer-free stent platform increased neointimal formation and inflammation following coronary stenting.

The effect of reactive oxygen species on whole blood aggregation and the endothelial cell-platelet interaction in patients with coronary heart disease.

BACKGROUND: The effect of reactive oxygen species (ROS) on platelet function in coronary heart disease (CHD) is complex and poorly defined. Platelet aggregation studies in healthy volunteers have demonstrated contrasting results when platelets are exposed to ROS. We investigated the effect of ROS on whole blood aggregation (WBA) and the endothelial cell-platelet interaction in patients with CHD. METHODS AND RESULTS: ROS generated by xanthine and xanthine oxidase caused a concentration-dependent inhibition of WBA in blood from healthy donors and patients with CHD. In patients with CHD, 100 µM xanthine and 100 mU/ml xanthine oxidase inhibited WBA in response to 3 µg/ml collagen by 28.9% (95% CI 15.9%-41.8%, p<0.001) and in response to 5 µM ADP by 36.0% (95% CI 9.6%-62.4%, p=0.005). Using nitrotyrosine expression, platelets isolated from patients with CHD were found to be susceptible to peroxynitrite damage. The addition of 1 × 10(5) cultured endothelial cells inhibited WBA in response to 3 µg/ml collagen by 31.2% (95% CI 12.2%-50.2%, p<0.05) and in response to 5 µM ADP by 31.6% (95% CI 2.5-60.7%, p<0.05). Addition of xanthine and xanthine oxidase did not alter this effect, however pre-treatment of endothelial cells with a nitric oxide synthase inhibitor (L-NAME) partly reversed the inhibition. CONCLUSION: ROS inhibit WBA in blood from patients with CHD. Whilst endothelial cells also inhibit WBA, the effect is attenuated by L-NAME, suggesting that nitric oxide is likely to remain an important protective mechanism against thrombosis in CHD.

Tissue localization of nanoparticles is altered due to hypoxia resulting in poor efficacy of curcumin nanoparticles in pulmonary hypertension.

The present study is an attempt to leverage therapeutic benefits of curcumin in pulmonary hypertension by encapsulating it in biodegradable poly(lactide-co-glycolic) acid nanoparticles. Pulmonary hypertension is induced in experimental animals by subjecting them to chronic hypoxic conditions. The ability of curcumin encapsulated nanoparticles to manage pulmonary hypertension is measured by right ventricular hypertrophy, haematocrit, vascular remodelling and target tissue levels of curcumin. Further, single oral dose tissue distribution of the nanoparticulate curcumin was also assessed under normoxic and hypoxic conditions. Orally administered nanoparticulate curcumin failed to offer any protection against hypoxia induced pulmonary hypertension as indicated by insignificant changes in right ventricular hypertrophy and vascular remodelling that are similar to untreated groups. A significant difference in the target tissue levels was observed between normoxic vs. hypoxic rats. The study suggests that hypoxia has a major role in the particle localization in lungs probably due to the altered blood flow, increased barrier properties of the lung vasculature and decreased endocytosis. The target tissue levels of curcumin under hypoxia are much lower to that achieved in normoxic rats probably due to difference in particle dynamics, resulting in the failure of treatment.

Threshold of peroxynitrite cytotoxicity in bovine pulmonary artery endothelial and smooth muscle cells.

Peroxynitrite is widely reported as highly cytotoxic; yet recent evidence indicates that at certain concentrations, it can induce pulmonary cell hyper-proliferation and tissue remodelling. This study aimed to establish the threshold concentration of peroxynitrite to induce functional impairment of bovine pulmonary artery endothelial (PAEC) and smooth muscle cells (PASMC). PAEC or PASMC were exposed to solution of peroxynitrite or 3-morpholinosydnonimine (SIN-1). Twenty-four hour cell viability, DNA synthesis, and protein biochemistry were assessed by trypan blue dye exclusion, [3H] thymidine incorporation and western blot analysis, respectively. Threshold concentration of peroxynitrite to significantly impair viability of PAEC and PASMC was 2 µM peroxynitrite. In PASMC and PAEC, low concentrations of peroxynitrite (2 nM-0.2 µM) increased cell proliferation and did not activate p38 MAP kinase. The decrease in DNA synthesis and cell viability caused by 2 µM peroxynitrite was associated with caspase-3 cleavage but not p38 activation. Also, 2-20 µM peroxynitrite significantly activated poly ADP ribose polymerase and stress activated kinase JNK in PAEC. However, the higher concentration of 20 µM peroxynitrite did cause a threefold increase in p38 activation. In conclusion, the threshold for the cytotoxic effects of peroxynitrite was 2 µM; which caused apoptotic cell death independent of p38 MAP kinase activation in pulmonary artery cells.

Acute hypoxia stimulates intracellular peroxynitrite formation associated with pulmonary artery smooth muscle cell proliferation.

There is separate evidence for peroxynitrite formation and hypoxia-induced cell proliferation in several models of hypoxic pulmonary hypertension. We therefore hypothesized that the stimulation of pulmonary artery smooth muscle cells (PASMCs) proliferation by hypoxia is due to peroxynitrite formation. The effect of hypoxia alone and in combination with ≤ 0.2 µM peroxynitrite on PASMCs was investigated in explants from bovine lungs grown in 1%, 5%, or 10% oxygen for 24 hours with or without peroxynitrite. At 0.1% fetal bovine serum, DNA synthesis of PASMCs (assessed by 3H thymidine incorporation) was increased by transient exposure to 0.2 µM peroxynitrite (by 158% ± 14%, P < 0.01) or to 24 hours of hypoxia (5% oxygen) (by 221% ± 17%, P < 0.01). Results were similar at 2.5% fetal bovine serum. Treatment of PASMCs with 0.2 µM peroxynitrite or 5% O2 hypoxia caused a significant increase in nitrotyrosine formation to a similar extent and intensity. The proliferative response to 0.2 µM peroxynitrite or to the combination of peroxynitrite plus 5% O2 was similar to the effect of 5% O2 alone and was abolished by simultaneous treatment with peroxynitrite scavenger-ebselen (5 µM). Our present data indicate that hypoxia can initiate peroxynitrite-induced proliferative events and suggest a mechanism for the vascular hypertrophy associated with pulmonary hypertension.

Tetrahydrobiopterin analogues with NO-dependent pulmonary vasodilator properties.

Reduced NO levels due to the deficiency of tetrahydrobiopterin (BH(4)) contribute to impaired vasodilation in pulmonary hypertension. Due to the chemically unstable nature of BH(4), it was hypothesised that oxidatively stable analogues of BH(4) would be able to support NO synthesis to improve endothelial dysfunction in pulmonary hypertension. Two analogues of BH(4), namely 6-hydroxymethyl pterin (HMP) and 6-acetyl-7,7-dimethyl-7,8-dihydropterin (ADDP), were evaluated for vasodilator activity on precontracted rat pulmonary artery rings. ADDP was administered to pulmonary hypertensive rats, followed by measurement of pulmonary vascular resistance in perfused lungs and eNOS expression by immunohistochemistry. ADDP and HMP caused significant relaxation in vitro in rat pulmonary arteries depleted of BH(4) with a maximum relaxation at 0.3µM (both P<0.05). Vasodilator activity of ADDP and HMP was completely abolished following preincubation with the NO synthase inhibitor, L-NAME. ADDP and HMP did not alter relaxation induced by carbachol or spermine NONOate. BH(4) itself did not produce relaxation. In rats receiving ADDP 14.1mg/kg/day, pulmonary vasodilation induced by calcium ionophore A23187 was augmented and eNOS immunoreactivity was increased. In conclusion, ADDP and HMP are two analogues of BH(4), which can act as oxidatively stable alternatives to BH(4) in causing NO-mediated vasorelaxation. Chronic treatment with ADDP resulted in improvement of NO-mediated pulmonary artery dilation and enhanced expression of eNOS in the pulmonary vascular endothelium. Chemically stable analogues of BH(4) may be able to limit endothelial dysfunction in the pulmonary vasculature.

Inhibition of inducible nitric oxide synthase promotes vein graft neoadventitial inflammation and remodelling.

BACKGROUND: Grafting veins into the arterial circulation causes endothelial damage and neointimal hyperplasia. However, the remodelling of vein grafts and the contribution of the endothelium is not well understood. Since nitric oxide (NO) has a crucial role in vascular function, we investigated the importance of NO synthases (NOSs) in vein graft re-endothelialization and remodelling in this study. METHODS AND RESULTS: Mouse isogenic vena cava was grafted into the carotid artery. Progressive remodelling of the grafted veins was evidenced by re-endothelialization at 2 weeks and subsequent appearance of vasomotor function at 4 weeks. Pharmacological inhibition of inducible NOS (iNOS) with the specific inhibitor 1400W, administered between 2 and 4 weeks after grafting, when re-endothelialization was complete, resulted in neoadventitial inflammation, neoadventitial thickening and impaired functional remodelling. CONCLUSION: Completion of re-endothelialization is pivotal in vein graft remodelling in the mouse and is associated with a series of changes in inflammation, proliferation and initiation of vascular functional remodelling. After re-endothelialization, iNOS upregulation may be an important mechanism to prevent secondary neoadventitial inflammation and preserve ongoing functional remodelling. iNOS activity could therefore be beneficial for long-term patency of the vein graft.

Modelling drug-eluting stents.

In this study, we consider a family of mathematical models to describe the elution of drug from polymer-coated stents into the arterial wall. Our models include the polymer layer, the media, the adventitia, a possible topcoat polymer layer and atherosclerotic plaque. We investigate the relative importance of transmural convection, diffusion and drug-dependent parameters in drug delivery and deposition. Furthermore, we investigate how the release rate from the stent can be altered and examine the resulting effect on cellular drug concentrations.

Protective effects of nanoparticulate coenzyme Q10 and curcumin on inflammatory markers and lipid metabolism in streptozotocin-induced diabetic rats: a possible remedy to diabetic complications.

Diabetes and its complications have been linked to increased levels of free radicals and systemic pro-inflammatory cytokines and to an altered lipid profile. Coenzyme Q10 and curcumin are potent antioxidants and anti-inflammatory agents but are underutilized clinically because of their poor bioavailability when administered orally. We have recently developed poly(D,L-lactic-co-glycolic acid)-based nanoparticles in which we have encapsulated coenzyme Q10 and curcumin to increase the oral bioavailability and therapeutic efficacy of the antioxidant molecules. These formulations when tested in streptozotocin-induced diabetic rats demonstrated protective effects on inflammatory markers as well as lipid metabolism. Coenzyme Q10 nanoparticulates reduced only C-reactive protein levels, whereas curcumin nanoparticles reduced levels of C-reactive protein, interleukin-6 and tumor necrosis factor-α. Administration of both nanoparticulates resulted in significant reductions of plasma triglycerides and total cholesterol and an increase in high-density lipoprotein cholesterol. Together, these data indicate the promise of coenzyme Q10 and curcumin in diabetes when delivered through nanoparticulate formulations.

Activation of prostanoid EP receptors by prostacyclin analogues in rabbit iliac artery: implications for anti-restenotic potential.

Prostacyclin analogues have the potential to be effective agents in a new generation of drug-eluting stents by virtue of prostanoid IP receptor mediated anti-proliferative effects on smooth muscle cells. However, prostanoid IP receptor mediated vessel relaxation is reduced at elevated analogue concentrations. The mechanisms underlying this loss of activity are unclear, and its influence on the anti-proliferative potential of these compounds remains to be determined. A classical organ bath approach was used to examine the functional response of the rabbit iliac artery to the prostacyclin analogues, AFP-07 and cicaprost. Selective receptor antagonists for prostanoid IP (RO-1138452), EP(1) (SC-51322) and EP(3) (L-798106) receptors were used to characterise the receptors involved. The effects of these agents on proliferation ([(3)H]-thymidine incorporation) of rabbit iliac artery smooth muscle cells stimulated by foetal calf serum were then studied. AFP-07 gave a bell-shaped log concentration-response curve consisting of prostanoid IP receptor mediated relaxation followed by reversal at higher concentrations. SC-51322 and L-798106 potentiated this relaxation, although only L-798106 completely removed the contractile element. The prostanoid EP(3) receptor agonist, sulprostone, produced constriction, which was attenuated by L-798106. RO-1138452 blocked the inhibitory action of AFP-07 and cicaprost on proliferation, implicating an involvement of prostanoid IP receptors. L-798106 had no effect on the anti-proliferative effect of cicaprost, but reduced the effect of AFP-07. Non-selective activation of prostanoid EP(3) receptors (and possibly prostanoid EP(1) receptors) compromises the relaxant activity of prostacyclin analogues, although it does not reduce the anti-proliferative capacity of these compounds in the model studied.

Pharmacotherapy to improve outcomes in infrainguinal bypass graft surgery: a review of current treatment strategies.

A total of 12,000 infrainguinal bypass grafts are performed annually in the United Kingdom, with outcomes suboptimal: 20% of above-knee vein grafts require intervention by 3 years. Transatlantic Inter-Society Consensus (TASC) guidelines exist on pharmacological management of peripheral vascular disease patients, however, little is recommended regarding optimum pharmacological management following revascularization to improve graft patency. The current recommendation is that all patients are on an antiplatelet agent following bypass grafting, the only intervention with significant evidence supporting use. This article will review pharmacological strategies aimed at improving the survival of infrainguinal vein grafts and the current evidence base for their use.

The sphingosine kinase inhibitor N,N-dimethylsphingosine inhibits neointimal hyperplasia.

BACKGROUND AND PURPOSE: Sphingosine-1-phosphate and its receptors may be involved in vascular smooth muscle cell (VSMC) proliferation following vascular injury. Here, we evaluate the effect of d-erythro-N,N-dimethylsphingosine (DMS), a sphingosine kinase (SK) inhibitor, on VSMC proliferation, apoptosis and neointimal formation. EXPERIMENTAL APPROACH: Growth responses in vitro to fetal calf serum (FCS) were measured by [(3)H]-thymidine incorporation and extracellular signal-regulated kinase-1/2 (ERK-1/2) activation in quiescent primary cultures of porcine VSMC in the presence and absence of various concentrations of the SK inhibitor DMS. In vivo treatment with DMS was delivered with a local endoluminal catheter, following balloon injury of coronary arteries. The artery intimal formation was investigated by angiography, myography and histomorphometry. KEY RESULTS: In vitro experiments indicated that DMS induced a dose-dependent reduction in [(3)H]-thymidine incorporation and ERK-1/2 activation via a protein kinase C (PKC) independent mechanism with an IC(50) value of 12 +/- 6 and 15 +/- 10 microM respectively. DMS also reduced Akt signalling. Four weeks following in vivo delivery of DMS, complete functional endothelial regeneration was observed in all treatment groups, with significant reduction of intimal formation (vehicle 23.7 +/- 4.6% vs. DMS infusion 8.92 +/- 2.9%, P < 0.05). CONCLUSIONS AND IMPLICATIONS: Taken together, these results demonstrate that local administration of the SK inhibitor, DMS, reduced neointimal formation, and this effect could involve inhibition of ERK-1/2 and Akt signalling, and modulation of smooth muscle growth.

Nitric oxide synthases in infants and children with pulmonary hypertension and congenital heart disease.

RATIONALE: Nitric oxide is an important regulator of vascular tone in the pulmonary circulation. Surgical correction of congenital heart disease limits pulmonary hypertension to a brief period. OBJECTIVES: The study has measured expression of endothelial (eNOS), inducible (iNOS), and neuronal nitric oxide synthase (nNOS) in the lungs from biopsies of infants with pulmonary hypertension secondary to cardiac abnormalities (n = 26), compared to a control group who did not have pulmonary or cardiac disease (n = 8). METHODS: eNOS, iNOS and nNOS were identified by immunohistochemistry and quantified in specific cell types. MEASUREMENTS AND MAIN RESULTS: Significant increases of eNOS and iNOS staining were found in pulmonary vascular endothelial cells of patients with congenital heart disease compared to control infants. These changes were confined to endothelial cells and not present in other cell types. Patients who strongly expressed eNOS also had strong expression of iNOS. CONCLUSION: Upregulation of eNOS and iNOS occurs at an early stage of pulmonary hypertension, and may be a compensatory mechanism limiting the rise in pulmonary artery pressure.

Understanding organic nitrates--a vein hope?

The organic nitrate drugs, such as glyceryl trinitrate (GTN; nitroglycerin), are clinically effective in angina because of their dilator profile in veins and arteries. The exact mechanism of intracellular delivery of nitric oxide (NO), or another NO-containing species, from these compounds is not understood. However, mitochondrial aldehyde dehydrogenase (mtALDH) has recently been identified as an organic nitrate bioactivation enzyme. Nitrate tolerance, the loss of effect of organic nitrates over time, is caused by reduced bioactivation and/or generation of NO-scavenging oxygen-free radicals. In a recent issue of the British Journal of Pharmacology, Wenzl et al. show that guinea-pigs, deficient in ascorbate, also have impaired responsiveness to GTN, but nitrate tolerance was not due to ascorbate deficiency that exhibited divergent changes in mtALDH activity. Thus, the complex function of mtALDH appears to be the key to activation of GTN, the active NO species formed and the induction of tolerance that can limit clinical effectiveness of organic nitrate drugs.

Inhibition of p38 MAPK reverses hypoxia-induced pulmonary artery endothelial dysfunction.

Hypoxia-induced endothelial dysfunction plays a crucial role in the pathogenesis of hypoxic pulmonary hypertension. p38 MAPK expression is increased in the pulmonary artery following hypoxic exposure. Recent evidence suggests that increased p38 MAPK activity is associated with endothelial dysfunction. However, the role of p38 MAPK activation in pulmonary artery endothelial dysfunction is not known. Sprague-Dawley rats were exposed to 2 wk hypobaric hypoxia, which resulted in the development of pulmonary hypertension and vascular remodeling. Endothelium-dependent relaxation of intrapulmonary vessels from hypoxic animals was impaired due to a reduced nitric oxide (NO) generation. This was despite increased endothelial NO synthase immunostaining and protein expression. Hypoxia exposure increased superoxide generation and p38 MAPK expression. The inhibition of p38 MAPK restored endothelium-dependent relaxation, increased bioavailable NO, and reduced superoxide production. In conclusion, the pharmacological inhibition of p38 MAPK was effective in increasing NO generation, reducing superoxide burden, and restoring hypoxia-induced endothelial dysfunction in rats with hypoxia-induced pulmonary hypertension. p38 MAPK may be a novel target for the treatment of pulmonary hypertension.

Selective arterial dilatation by glyceryl trinitrate is not associated with nitric oxide formation in vitro.

AIMS: Glyceryl trinitrate (GTN) is the most commonly used anti-anginal agent, yet its mechanism of action has still to be fully established. Release of nitric oxide (NO) and the selectivity of GTN in the venous system are believed to be crucial to this drug's anti-anginal action. METHODS: Rat superior mesenteric arteries and renal veins were mounted in a wire myograph with an intraluminal NO microsensor. RESULTS: In the superior mesenteric arteries, GTN (1 nM to 10 microM) produced a dose-dependent vasodilatation without NO release, except at concentrations supramaximal for relaxation. GTN was found to be markedly less potent in a wide range of veins tested, and lowering the oxygen concentrations in the myograph to that of the venous system did not improve the venodilator activity of GTN. CONCLUSION: This is the first time that NO release from GTN has been monitored electrochemically in real time, simultaneously with vasodilatation. Unlike the endothelium-dependent vasodilator carbachol, NO could only be measured at concentrations of GTN that are supramaximal for relaxation. GTN was found to be arterioselective in vitro, even when oxygen levels were lowered to mimic those of the venous system in vivo.