George R. Thomas

Tolerance to nitroglycerin-induced preconditioning of the endothelium: a human in vivo study

Damage and dysfunction of the vascular endothelium critically influence clinical outcomes after ischemia and reperfusion (I/R). Brief exposure to organic nitrates can protect the vascular endothelium from I/R injury via a mechanism that is similar to ischemic preconditioning and is independent of hemodynamic changes. The clinical relevance of these protective effects clearly depends on whether they can be sustained over time. Twenty-four healthy (age 25-32) male volunteers were randomized to receive 1) transdermal nitroglycerin (GTN; 0.6 mg/h) administered for 2 h on 1 day only, 2) transdermal GTN for 2 h/day for 7 days, or 3) continuous therapy with transdermal GTN for 7 days. Eight volunteers underwent continuous GTN therapy followed by intra-arterial infusion of the antioxidant vitamin C. Finally, five additional subjects underwent no therapy and served as controls. Endothelial function measurements were performed before and after induction of I/R of the arm. I/R caused a significant blunting of the flow responses to acetylcholine in the control group (P < 0.01 vs. before I/R). A single 2-h GTN dosage, given 24 h before I/R, prevented I/R-induced endothelial dysfunction [P = not significant (NS) vs. before I/R], but this protective effect was completely lost after 1 wk of GTN administration 2 h/day (P < 0.05 vs. before I/R; P = NS vs. control). In subjects who received continuous GTN, endothelial responses were blunted before I/R, and I/R did not cause further endothelial dysfunction. Finally, vitamin C normalized acetylcholine responses and prevented the loss of preconditioning associated with prolonged GTN. In a separate experimental model using isolated human endothelial cells, short-term incubation with GTN caused upregulation of heme oxygenase, an effect that was lost after prolonged GTN administration. Although a single administration of GTN is able to protect the endothelium from I/R-induced endothelial dysfunction, this protection is lost upon prolonged exposure, likely via an oxidative mechanism.

Nitroglycerin Attenuates Human Endothelial Progenitor Cell Differentiation, Function, and Survival

Endothelial progenitor cells (EPCs) participate in angiogenesis and the response to chronic ischemia. Risk factors and cardiovascular disease attenuate EPC number, function, and survival. Continuous therapy with nitroglycerin (glyceryl trinitrate; GTN) is associated with increased vascular oxidative stress, leading to nitrate tolerance and endothelial dysfunction. Thus, GTN therapy may also affect EPCs. The purpose of this study was to determine whether continuous exposure to GTN in vivo or during ex vivo expansion affects the circulating number and functional characteristics of human EPCs. To determine the effects of continuous in vivo GTN exposure, EPCs isolated from 28 healthy males before and after receiving 0.6 mg/h GTN (n = 17) or no treatment (n = 11) for 1 week were expanded for 6 days and compared. To determine the effects of continuous ex vivo GTN exposure, EPCs isolated before randomization were expanded for 6 days in medium supplemented with 100 nM, 300 nM, or 1 μM GTN. EPCs expanded without GTN served as controls (n = 10). In vivo, GTN exposure significantly increased the percentage of circulating cells expressing the EPC marker CD34 and increased the susceptibility of expanded EPCs to apoptosis but had no impact on the phenotypic differentiation or migration of EPCs. Ex vivo, GTN exposure increased apoptosis while decreasing phenotypic differentiation, migration, and mitochondrial dehydrogenase activity of EPCs, compared with EPCs expanded in the absence of GTN. Taken together, these results suggest that continuous GTN therapy might impair EPC-mediated processes, an effect that could be detrimental in the setting of ischemic cardiovascular disease.

Standard versus low-dose transdermal nitroglycerin: differential effects on the development of tolerance and abnormalities of endothelial function.

We compared standard (0.6 mg/h) versus low-dose (0.05 mg/h) transdermal nitroglycerin (TGTN) on acute hemodynamic parameters, the development of tolerance, and endothelial function. Study 1 randomized six healthy volunteers to receive 0.6 mg/h or 0.05 mg/h TGTN in a crossover design study (6-day washout period) with measurements of heart rate, blood pressure, radial artery waveforms, and aortic augmentation index taken at baseline and 1, 2, and 3 hours after initial TGTN application. Study 2 enrolled 24 healthy volunteers to receive 400 μg of sublingual nitroglycerin spray followed by 400 μg of inhaled salbutamol 90 minutes later. Measurements of heart rate, blood pressure, radial artery waveforms as well as aortic augmentation index were taken at baseline and at 5, 10, and 20 minutes after each treatment. They were randomized to either 0.6 mg/h or 0.05 mg/h of TGTN, and the same measurements were repeated after 6 days. In Study 1, there was no significant difference in the response to both doses (analysis of variance, P < 0.05). In Study 2, the decrease in aortic augmentation index in response to sublingual nitroglycerin and salbutamol was attenuated after sustained therapy with 0.6 mg/h of TGTN (versus 0.05 mg/h, P < 0.05). This investigation documents that 0.05 mg/h TGTN has identical acute hemodynamic effects compared with 0.6-mg/h dose without causing tolerance or endothelial dysfunction.

Pyridoxal-5′-phosphate (MC-1), a vitamin B6 derivative, inhibits expressed P2X receptors

P2X receptors are cation-permeable ligand-gated ion channels that open in response to the binding of ATP. These receptors are present in many excitable cells, including neurons, striated muscle cells, epithelial cells, and leukocytes. They mediate fast excitatory neurotransmission in the central and peripheral nervous systems and are thought to be involved in neuropathic pain, inflammation, and cell damage following ischemia-reperfusion injuries. P2X receptors are thus a target for the development of new therapeutics to treat chronic pain and inflammation. In this study, we characterized the inhibition caused by pyridoxal-5-phosphate, a natural metabolite of vitamin B6 (MC-1), of P2X₂, P2X₄, P2X₇, and P2X₂/₃ receptors stably expressed in HEK293 cells using the patch-clamp technique in the whole-cell configuration. We also tested a new approach using VC6.1, a modified cameleon calcium-sensitive fluorescent protein, to characterize the inhibition of P2X₂ and P2X₂/₃. MC-1 blocked these two P2X receptors, with an IC₅₀ of 7 and 13 μmol/L, respectively. P2X₂ exhibited the highest affinity for VC6.1, and the chimeric receptor P2X₂/₃, the lowest. The patch-clamp and imaging approaches gave similar results and indicated that VC6.1 may be useful for high throughput drug screening. Pyridoxal-5-phosphate is an efficient P2X blocker and can be classified as a P2X antagonist.

Daily low-dose folic acid supplementation does not prevent nitroglycerin-induced nitric oxide synthase dysfunction and tolerance: A human in vivo study

INTRODUCTIONnContinuous treatment with nitroglycerin (GTN) causes tolerance and endothelial dysfunction, both of which may involve endothelial nitric oxide synthase (eNOS) dysfunction. eNOS dysfunction may be linked to depletion of tetrahydrobiopterin, and folic acid may be involved in the regeneration of this cofactor. It has been demonstrated that 10 mg⁄day folic acid supplementation prevents the development of GTN tolerance and GTN-induced endothelial dysfunction. However, the efficacy of daily lower-dose folic acid supplementation for preventing these phenomena has not been investigated.nnnOBJECTIVEnTo determine the effect of 1 mg⁄day folic acid supplementation on responses to sustained GTN therapy.nnnMETHODS AND RESULTSnOn visit 1, 20 healthy male volunteers were randomly assigned to receive either oral folic acid (1 mg⁄day) or placebo for one week in a double- blind study. All subjects also received continuous transdermal GTN (0.6 mg⁄h). On visit 2, forearm blood flow was measured using venous occlusion strain-gauge plethysmography in response to incremental intra-arterial infusions of acetylcholine, N-monomethyl-L-arginine and GTN. Subjects in both groups displayed significantly decreased responses to acetylcholine and N-monomethyl-L-arginine infusions compared with a control group that received no treatment. Responses to GTN were also significantly diminished in both groups (P<0.05 for all).nnnDISCUSSIONnThe present data demonstrate that daily supplementation with 1 mg folic acid does not prevent the development of GTN-induced eNOS dysfunction or tolerance.

MECHANISM AND LOCATION OF ADENOSINE-INDUCED PULMONARY VEIN-LEFT ATRIAL RECONNECTION FOLLOWING PULMONARY VEIN ISOLATION

Results: In 9 (30%) pts, ADO-induced PV conduction occurred in 13/106 (12%) PVs. Transient ADO-induced PV conduction was seen with 10 PVs (mean duration 14 ± 5 s) while sustained PV reconnection was seen with 3 PVs. Onset of ADO-induced PV conduction (mean 13 ± 6 s post bolus) occurred prior to sinus slowing or AV block in 7 (54%) PVs, during sinus slowing and/or AV block in 6 (46%) PVs, and in none during the sinus tachycardia phase. PV reconnection was most commonly seen at the left atrial appendage-PV ridge of the left superior PV (23% of all reconnected PVs).

DIFFERENTIATING PACEMAKER-MEDIATED TACHYCARDIA FROM PACEMAKER ATRIAL TRACKING DURING WIDE COMPLEX TACHYCARDIA: UTILITY OF V-A-A-V VS. V-A-V RESPONSE AFTER PVARP EXTENSION

Background: Dual chamber pacemakers can lead to two forms of pacemaker-facilitated tachycardia -pacemaker-mediated tachycardia (PMT) and tracking of sinus or atrial tachycardia. Current pacemaker algorithms are not always able to properly classify them. We introduce a new algorithm for differentiating these particular mechanisms of pacemaker-facilitated tachycardia, which is based on the specific termination response to post-ventricular atrial refractory period (PVARP) extension (see figure).