Growth of algal biomass in laboratory and in large-scale algal photobioreactors in the temperate climate of western Germany.

Growth of Chlorella vulgaris was characterized as a function of irradiance in a laboratory turbidostat (1L) and compared to batch growth in sunlit modules (5-25L) of the commercial NOVAgreen photobioreactor. The effects of variable sunlight and culture density were deconvoluted by a mathematical model. The analysis showed that algal growth was light-limited due to shading by external construction elements and due to light attenuation within the algal bags. The model was also used to predict maximum biomass productivity. The manipulative experiments and the model predictions were confronted with data from a production season of three large-scale photobioreactors: NOVAgreen (<36,000L), IGV (2,500-3,500L), and Phytolutions (28,000L). The analysis confirmed light-limitation in all three photobioreactors. An additional limitation of the biomass productivity was caused by the nitrogen starvation that was used to induce lipid accumulation. Reduction of shading and separation of biomass and lipid production are proposed for future optimization.

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomyces cerevisiae.

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.

Endothelin-1 is a key mediator of coronary vasoconstriction in patients with transplant coronary arteriosclerosis.

BACKGROUND: Transplant coronary arteriosclerosis (TCA) is the principal long-term complication in cardiac transplant recipients. The mediators responsible for vascular proliferation and vasoconstriction typical of TCA remain largely unknown. We tested whether endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, contributes to the pathogenesis and manifestations of TCA. METHODS AND RESULTS: BQ-123, an ET-1 receptor-A antagonist, was infused into a coronary artery (40 nmol/min for 60 minutes) of 18 subjects, 6 + or - 4 years after transplantation. Vasomotor responses were measured in the infused artery and in a noninfused control artery in patients with (n=10) and without (n=8) advanced TCA (108 total coronary segments). Changes in diameters were compared at 15-minute intervals up to 60 minutes. Contribution of ET-1 to coronary constrictor tone was assessed by comparing vasodilation from BQ-123 with that of the maximal vasodilator nitroglycerin (200-microg intracoronary bolus). BQ-123 dilated coronary arteries of transplanted patients (8.4% at 60 minutes versus -0.4% in noninfused arteries, P<0.001). Dilation was greater for arteries with advanced TCA defined as diameter stenosis > or = 15% (dilation 15.2% with versus 0.6% without advanced TCA, P=0.004). Judged against the response to nitroglycerin, ET-1 accounted for 53.2% of coronary tone in advanced TCA but only 12.9% without advanced TCA. CONCLUSIONS: This study shows for the first time in humans that ET-1 is an important mediator of coronary vasoconstriction in TCA and accounts for >50% of the increased vasomotor tone. Therapeutic targeting of ET-1 may retard the development of TCA.

Impact of obstructive sleep apnea on the occurrence of restenosis after elective percutaneous coronary intervention in ischemic heart disease.

RATIONALE: There is growing evidence that obstructive sleep apnea is associated with coronary artery disease. However, there are no data on the course of coronary stenosis after percutaneous coronary intervention in patients with obstructive sleep apnea. OBJECTIVES: To determine whether sleep apnea is associated with increased late lumen loss and restenosis after percutaneous coronary intervention. METHODS: 78 patients with coronary artery disease who underwent elective percutaneous coronary intervention were divided in 2 groups: 43 patients with an apnea hypopnea - Index < 10/h (group I) and 35 pt. with obstructive sleep apnea and an AHI > 10/h (group II). Late lumen loss, a marker of restenosis, was determined using quantitative coronary angiography after 6.9 +/- 3.1 months. MAIN RESULTS: Angiographic restenosis (>50% luminal diameter), was present in 6 (14%) of group I and in 9 (25%) of group II (p = 0.11). Late lumen loss was significant higher in pt. with an AHI > 10/h (0.7 +/- 0.69 mm vs. 0.38 +/- 0.37 mm, p = 0.01). Among these 35 patients, 21(60%) used their CPAP devices regularly. There was a marginally lower late lumen loss in treated patients, nevertheless, this difference did not reach statistical significance (0.57 +/- 0.47 mm vs. 0.99 +/- 0.86 mm, p = 0.08). There was no difference in late lumen loss between treated patients and the group I (p = 0.206). CONCLUSION: In summary, patients with OSA and coronary artery disease have a higher degree of late lumen loss, which is a marker of restenosis and vessel remodeling after elective percutaneous intervention.

Impact of coronary endothelial function on the progression of cardiac transplant-associated arteriosclerosis: effect of anti-oxidant vitamins C and E.

BACKGROUND: Excessive vascular oxidant stress has been implicated in cardiac transplant-associated arteriosclerosis (TxAA). In a recent placebo-controlled study of 40 cardiac transplant recipients, vitamin C 500 mg twice a day and vitamin E 400 IU twice a day for 1 year retarded the progression of TxAA, as assessed by intravascular ultrasound (IVUS). Endothelial dysfunction is a key feature of TxAA and reflects oxidant stress. We hypothesized that coronary endothelial dysfunction portends greater TxAA progression and a larger therapeutic response to anti-oxidant vitamins. METHODS: In this pre-specified analysis, the 40 cardiac transplant recipients were categorized according to normal or abnormal coronary endothelial vasomotor function at baseline, as assessed by acetylcholine (10(-8) to 10(-6) mol/liter). The effect of anti-oxidant vitamins within these two groups of patients was assessed by the change in intimal index over 1 year using IVUS. RESULTS: With placebo (n = 21), the increase in intimal index was greater in the presence vs absence of endothelial dysfunction (11 +/- 3% vs 5 +/- 1%, p < 0.05). Among patients with endothelial dysfunction (n = 21), the intimal index increased 11 +/- 3% with placebo, but decreased -1 +/- 2% with vitamins (p = 0.002). Among patients with normal endothelial function (n = 14), the intimal index increased 5 +/- 1% with placebo and 1 +/- 1% with vitamins (p < 0.05). CONCLUSIONS: Endothelial dysfunction indicates rapid TxAA progression, even in the statin era. Although anti-oxidant vitamins reduce disease progression in patients with normal or abnormal endothelial function, the magnitude of benefit is larger in patients with endothelial dysfunction.

Long-term effect of combined vitamins E and C on coronary and peripheral endothelial function.

OBJECTIVES: We tested whether long-term administration of antioxidant vitamins C and E improves coronary and brachial artery endothelial function in patients with coronary artery disease (CAD). BACKGROUND: Endothelial function is a sensitive indicator of vascular health. Oxidant stress and oxidized low-density lipoprotein (LDL) impair endothelial function by reducing nitric oxide bioavailability in the artery wall. METHODS: We randomly assigned 30 subjects with CAD to combined vitamin E (800 IU per day) and C (1000 mg per day) or to placebos in a double-blind trial. Coronary artery endothelial function was measured as the change in coronary artery diameter to acetylcholine infusions (n = 18 patients), and brachial artery endothelial function was assessed by flow-mediated dilation (n = 25 patients) at baseline and six months. Plasma markers of oxidant stress (oxidized LDL and autoantibodies) were also measured. RESULTS: Plasma alpha-tocopherol (p < 0.001) and ascorbic acid (p < 0.02) increased with active therapy. Compared to placebo, there was no improvement in coronary and brachial endothelial vasomotor function over six months. Although vitamins C and E tended to reduce F2-isoprostanes (p = 0.065), they failed to alter oxidized LDL or autoantibodies to oxidized LDL. CONCLUSIONS: Long-term oral vitamins C and E do not improve key mechanisms in the biology of atherosclerosis or endothelial dysfunction, or reduce LDL oxidation in vivo.

No-reflow is an independent predictor of death and myocardial infarction after percutaneous coronary intervention.

BACKGROUND: No-reflow occurring during percutaneous coronary intervention (PCI) has been associated with poor inhospital outcomes. The objectives of this analysis were to evaluate the occurrence of no-reflow as an independent predictor of adverse events and to determine whether treatment with intracoronary vasodilator therapy affected clinical outcomes. METHODS: We prospectively collected data from 4264 consecutive patients undergoing PCI, identifying those with no-reflow, and analyzed their treatments and clinical outcomes. RESULTS: No-reflow was identified in 135 of 4264 patients (3.2%). Baseline demographics were comparable, but patients with no-reflow were more likely to have acute myocardial infarction, unstable angina, and cardiogenic shock and to have undergone saphenous vein graft interventions. No-reflow was highly predictive of postprocedural myocardial infarction (17.7% vs 3.5% in patients without no-reflow, P <.001) and death (7.4% vs 2.0%, P <.001) and remained a strong independent predictor of death or myocardial infarction after multivariate analysis (odds ratio 3.6, P <.001). The administration of intracoronary verapamil, sodium nitroprusside, or both was not associated with a reduction in the rate of death or myocardial infarction (adjusted odds ratio of death or myocardial infarction 1.04, P =.945 for nitroprusside; and adjusted odds ratio of death or myocardial infarction 0.94, P =.91 for verapamil), despite an improvement in angiographic flow rates for patients treated with sodium nitroprusside. CONCLUSIONS: No-reflow is a strong independent predictor of inhospital mortality and postprocedural myocardial infarction. Administration of verapamil or sodium nitroprusside was not associated with improved inhospital outcomes in patients with no-reflow, although anterograde flow rates were improved in patients treated with sodium nitroprusside.

Endothelial function. From vascular biology to clinical applications.

The endothelium, by releasing nitric oxide (NO), promotes vasodilation and inhibits inflammation, thrombosis, and vascular smooth muscle cell proliferation. These biological actions of NO make it an important component in the endogenous defense against atherosclerosis and its overt clinical complications. Loss of the functional integrity of the endothelium, as seen commonly in the milieu of cardiovascular risk factors, plays an integral role in all stages of atherosclerosis from lesion initiation to plaque rupture. A number of established techniques can assess endothelial function in human vascular beds. The outcome of endothelial testing has profound prognostic implications and is an independent predictor of atherosclerosis disease progression and cardiovascular event rates. The large clinical benefit of statins and angiotensin-converting enzyme inhibitors in patients with atherosclerosis involves favorable effects of endothelial function. Studies of endothelial function represent a prime example of a successful application of insights derived from vascular biology at the bedside.

Circulating autoantibodies to oxidized LDL correlate with impaired coronary endothelial function after cardiac transplantation.

OBJECTIVE: The oxidative modification of low density lipoprotein (LDL) may play a role in the pathogenesis of transplant-associated arteriosclerosis. Oxidized LDL (OxLDL) is immunogenic as well as atherogenic, and the level of autoantibodies to OxLDL has been taken as an index of the oxidant state of LDL. Because endothelial dysfunction is key in the initiation of transplant-associated arteriosclerosis, we postulated that the level of OxLDL autoantibody is associated with the degree of impairment of coronary endothelial function. METHODS AND RESULTS: Coronary endothelium-dependent dilation was assessed by using intracoronary acetylcholine and endothelium-independent dilation by nitroglycerin in 36 cardiac transplant recipients within 1 year of transplantation. The coronary responses to acetylcholine ranged from -37% (vasoconstriction) to 31% (vasodilation), and the responses to nitroglycerin ranged from 0% to 42% (vasodilation). The coronary vasomotor response to acetylcholine was significantly and inversely related to OxLDL autoantibody levels (r=-0.43, P<0.01) but not LDL levels (r=-0.04, P=0.83) or circulating OxLDL levels detected by monoclonal antibody EO6 (r=-0.27, P=0.11). The coronary artery response to nitroglycerin was not related to levels of OxLDL autoantibodies, LDL, or EO6 (all P=NS). CONCLUSIONS: Autoantibodies to OxLDL are increased in patients with coronary endothelial dysfunction in the first year after cardiac transplantation. The oxidative modification of LDL by inducing endothelial dysfunction in cardiac transplant recipients may be a critical step in the atherogenic effects of LDL and may provide a potential target for therapy.

Effect of vitamins C and E on progression of transplant-associated arteriosclerosis: a randomised trial.

BACKGROUND: Cardiac transplantation is associated with oxidant stress, which may contribute to the development of accelerated coronary arteriosclerosis. We postulated that treatment with antioxidant vitamins C and E would retard the progression of transplant-associated arteriosclerosis. METHODS: In a double-blind prospective study, 40 patients (0-2 years after cardiac transplantation) were randomly assigned vitamin C 500 mg plus vitamin E 400 IU, each twice daily (n=19), or placebo (n=21) for 1 year. The primary endpoint was the change in average intimal index (plaque area divided by vessel area) measured by intravascular ultrasonography (IVUS). Coronary endothelium-dependent vasoreactivity was assessed with intracoronary acetylcholine infusions. IVUS, coronary vasoreactivity, and vitamin C and E plasma concentrations were assessed at baseline and at 1 year follow-up. All patients received pravastatin. Analyses were by intention to treat. FINDINGS: Vitamin C and E concentrations increased in the vitamin group (vitamin C 43 [SD 21] to 103 [43] mmol/L; vitamin E 24 [14] to 65 [27] mmol/L) but did not change in the placebo group (vitamin C 45 [15] vs 43 [16] mmol/L; vitamin E 27 [14] vs 27 [9] mmol/L; p<0.0001 for difference between groups). During 1 year of treatment, the intimal index increased in the placebo group by 8% (SE 2) but did not change significantly in the treatment group (0.8% [1]; p=0.008). Coronary endothelial function remained stable in both groups. INTERPRETATION: Supplementation with antioxidant vitamins C and E retards the early progression of transplant-associated coronary arteriosclerosis.