Naris Thengchaisri

Ischemia-reperfusion selectively impairs nitric oxide-mediated dilation in coronary arterioles: counteracting role of arginase

A reduction in L‐arginine availability has been implicated in the impairment of endothelium‐ dependent nitric oxide (NO)‐mediated vasodilation by ischemia‐reperfusion (I/R). However, the mechanisms contributing to dysregulation of the L‐arginine pool remain unknown. Because endothelial cells can metabolize L‐arginine via two major enzymes, that is, NO synthase (NOS) and arginase, we hypothesized that up‐regulation of arginase during I/R reduces L‐arginine availability to NOS and thus impairs NO‐mediated vasodilation. To test this hypothesis, a local I/R was produced in the porcine heart by occlusion of a small branch of left anterior descending artery for 30 min, followed by reperfusion for 90 min. Arterioles (60−110 µm) isolated from non‐ischemic and ischemic regions of subepicardium were cannulated and pressurized without flow for in vitro study. Vessels from both regions developed similar levels of basal tone. Although the dilation of I/R vessels to endothelium‐independent agonist sodium nitroprusside was not altered, the endothelium‐dependent NO‐mediated dilations to adenosine and serotonin were attenuated. I/R not only inhibited arteriolar production of NO but also increased arteriolar arginase activity. Arginase inhibitor α‐difluoromethylornithine enhanced NO production/dilation in normal vessels and also restored the NO‐mediated function in I/R vessels. Treating I/R vessels with L‐arginine also restored vasodilations. Immunohistochemical data revealed that I/R up‐ regulated arginase but down‐regulated NOS expression in the arteriolar endothelium. Pretreating the animals with protein synthesis inhibitor cycloheximide prevented I/R‐induced arginase up‐ regulation and also preserved NO‐mediated vascular function. These results suggest that one mechanism by which I/R inhibits NO‐mediated arteriolar dilation is through increased arginase activity, which limits the availability of L‐arginine to NOS for NO production. In addition, the inability of arginase blockade or L‐arginine supplementation to completely restore vasodilatory function may be attributable to the down‐regulation of endothelial NOS expression.

Upregulation of Arginase by H2O2 Impairs Endothelium-Dependent Nitric Oxide-Mediated Dilation of Coronary Arterioles

Objective—Overproduction of reactive oxygen species such as hydrogen peroxide (H2O2) has been implicated in various cardiovascular diseases. However, mechanism(s) underlying coronary vascular dysfunction induced by H2O2 is unclear. We studied the effect of H2O2 on dilation of coronary arterioles to endothelium-dependent and endothelium-independent agonists. Methods and Results—Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. All vessels developed basal tone and dilated dose-dependently to activators of nitric oxide (NO) synthase (adenosine and ionomycin), cyclooxygenase (arachidonic acid), and cytochrome P450 monooxygenase (bradykinin). Intraluminal incubation of vessels with H2O2 (100 &mgr;mol/L, 60 minutes) did not alter basal tone but inhibited vasodilations to adenosine and ionomycin in a manner similar as that by NO synthase inhibitor L-NAME. H2O2 affected neither endothelium-dependent responses to arachidonic acid and bradykinin nor endothelium-independent dilation to sodium nitroprusside. The inhibited adenosine response was not reversed by removal of H2O2 but was restored by excess L-arginine. Inhibition of L-arginine consuming enzyme arginase by α-difluoromethylornithine or N&ohgr;-hydroxy-nor-l-arginine also restored vasodilation. Administering deferoxamine, an inhibitor of hydroxyl radical production, prevented the H2O2-induced impairment of vasodilation to adenosine. Western blot and reverse-transcription polymerase chain reaction results indicated that arginase I was upregulated after treating vessels with H2O2. Conclusions—H2O2 specifically impairs endothelium-dependent NO-mediated dilation of coronary microvessels by reducing L-arginine availability through upregulation of arginase. The formation of hydroxyl radicals from H2O2 may contribute to this process.

Selective Activation of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Mediates C-Reactive Protein–Evoked Endothelial Vasodilator Dysfunction in Coronary Arterioles

Rationale: Studies in cultured endothelium implicate that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) or Fc&ggr; receptor II (CD32) contributes to the proatherogenic effects of C-reactive protein (CRP). However, the identity of the receptors linking to deleterious actions of CRP in vasomotor regulation remains unknown. Objective: We tested the hypothesis that LOX-1 contributes to adverse effects of CRP on endothelium-dependent vasomotor function in resistance arterioles. Methods and Results: Porcine coronary arterioles were isolated for vasoreactivity study, dihydroethidium fluorescence staining of superoxide, immunohistochemical localization of receptors, immunoprecipitation of receptor/CRP interaction, and protein blot. Intraluminal treatment of pressurized arterioles with a pathophysiological level of CRP (7 µg/mL; 60 minutes) attenuated endothelium-dependent nitric oxide–mediated and prostacyclin-mediated dilations to serotonin and arachidonic acid, respectively. LOX-1 and CD32 were detected in the endothelium of arterioles. Blockade of LOX-1 with either pharmacological antagonist &kgr;-carrageenan or anti–LOX-1 antibody prevented the detrimental effect of CRP on vasodilator function, whereas anti-CD32 antibody treatment was ineffective. Denudation of endothelium and blockade of LOX-1 but not CD32 prevented CRP-induced elevation of superoxide in the vessel wall. CRP was coimmunoprecipitated with LOX-1 and CD32 from CRP-treated arterioles. Similarly, LOX-1 and CD32 blockade prevented CRP-induced arteriolar expression of plasminogen activator inhibitor-1, a thrombogenic protein. Conclusions: CRP elicits endothelium-dependent oxidative stress and compromises nitric oxide–mediated and prostacyclin-mediated vasomotor function via LOX-1 activation. In contrast, both LOX-1 and CD32 mediate plasminogen activator inhibitor-1 upregulation in arterioles by CRP. Thus, activation of LOX-1 and CD32 may contribute to vasomotor dysfunction and proatherogenic actions of CRP, respectively.

Selective Activation of LOX-1 Mediates C-Reactive Protein Evoked Endothelial Vasodilator Dysfunction in Coronary Arterioles

Rationale: Studies in cultured endothelium implicate that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) or Fc&ggr; receptor II (CD32) contributes to the proatherogenic effects of C-reactive protein (CRP). However, the identity of the receptors linking to deleterious actions of CRP in vasomotor regulation remains unknown. Objective: We tested the hypothesis that LOX-1 contributes to adverse effects of CRP on endothelium-dependent vasomotor function in resistance arterioles. Methods and Results: Porcine coronary arterioles were isolated for vasoreactivity study, dihydroethidium fluorescence staining of superoxide, immunohistochemical localization of receptors, immunoprecipitation of receptor/CRP interaction, and protein blot. Intraluminal treatment of pressurized arterioles with a pathophysiological level of CRP (7 µg/mL; 60 minutes) attenuated endothelium-dependent nitric oxide–mediated and prostacyclin-mediated dilations to serotonin and arachidonic acid, respectively. LOX-1 and CD32 were detected in the endothelium of arterioles. Blockade of LOX-1 with either pharmacological antagonist &kgr;-carrageenan or anti–LOX-1 antibody prevented the detrimental effect of CRP on vasodilator function, whereas anti-CD32 antibody treatment was ineffective. Denudation of endothelium and blockade of LOX-1 but not CD32 prevented CRP-induced elevation of superoxide in the vessel wall. CRP was coimmunoprecipitated with LOX-1 and CD32 from CRP-treated arterioles. Similarly, LOX-1 and CD32 blockade prevented CRP-induced arteriolar expression of plasminogen activator inhibitor-1, a thrombogenic protein. Conclusions: CRP elicits endothelium-dependent oxidative stress and compromises nitric oxide–mediated and prostacyclin-mediated vasomotor function via LOX-1 activation. In contrast, both LOX-1 and CD32 mediate plasminogen activator inhibitor-1 upregulation in arterioles by CRP. Thus, activation of LOX-1 and CD32 may contribute to vasomotor dysfunction and proatherogenic actions of CRP, respectively.

Regulation of Coronary Vasomotor Function by Reactive Oxygen Species

Overproduction of reactive oxygen species is closely associated with cardiovascular diseases in part by impairing endothelial function and consequently compromising blood flow regulation. Superoxide and hydrogen peroxide are elevated under various disease states with reduced endothelium-derived nitric oxide bioavailability. The oxidative stress elicited by angiotensin II, C-reactive protein and tumor necrosis factor-α is mediated by the activation of different redox signaling pathways in the microvasculature. The upregulation of L-arginine consuming enzyme arginase also contributes to the reduced nitric oxide bioavailability during oxidative stress. Hydrogen peroxide exhibits vasodilator function in the coronary microcirculation and plays an important role in the physiological regulation of coronary blood flow. However, excessive production of hydrogen peroxide impairs endothelial function by reducing L-arginine availability through hydroxyl radical-mediated upregulation of arginase. This review summarizes the current knowledge on the effects superoxide and hydrogen peroxide on vasomotor function regulated by the endothelium-derived nitric oxide and prostacyclin in the coronary microcirculation.

Endothelin-1 impairs coronary arteriolar dilation: Role of p38 kinase-mediated superoxide production from NADPH oxidase

Elevated levels of endothelin-1 (ET-1), a potent vasoactive peptide, are implicated as a risk factor for cardiovascular diseases by exerting vasoconstriction. The aim of this study was to address whether ET-1, at sub-vasomotor concentrations, elicits adverse effects on coronary microvascular function. Porcine coronary arterioles (50-100μm) were isolated, cannulated and pressurized without flow for in vitro study. Diameter changes were recorded using a videomicrometer. Arterioles developed basal tone (60±3μm) and dilated to the endothelium-dependent nitric oxide (NO)-mediated vasodilators serotonin (1nmol/L to 0.1μmol/L) and adenosine (1nmol/L to 10μmol/L). Treating the vessels with a clinically relevant sub-vasomotor concentration of ET-1 (10pmol/L, 60min) significantly attenuated arteriolar dilations to adenosine and serotonin but not to endothelium-independent vasodilator sodium nitroprusside. The arteriolar wall contains ETA receptors and the adverse effect of ET-1 was prevented by ETA receptor antagonist BQ123, the superoxide scavenger Tempol, the NADPH oxidase inhibitors apocynin and VAS2870, the NOX2-based NADPH oxidase inhibitor gp91 ds-tat, or the p38 kinase inhibitor SB203580. However, ETB receptor antagonist BQ788, H2O2 scavenger catalase, scrambled gp91 ds-tat, or inhibitors of xanthine oxidase (allopurinol), PKC (Gö 6983), Rho kinase (Y27632), and c-Jun N-terminal kinase (SP600125) did not protect the vessel. Immunohistochemical staining showed that ET-1 elicited Tempol-, apocynin- and SB203580-sensitive superoxide productions in the arteriolar wall. Our results indicate that exposure of coronary arterioles to a pathophysiological, sub-vasomotor concentration of ET-1 leads to vascular dysfunction by impairing endothelium-dependent NO-mediated dilation via p38 kinase-mediated production of superoxide from NADPH oxidase following ETA receptor activation.

Association of Ehrlichia canis, Hemotropic Mycoplasma spp. and Anaplasma platys and severe anemia in dogs in Thailand

Canine tick-borne bacteria; Ehrlichia canis, hemotropic Mycoplasma spp. and Anaplasma spp., are organisms transmitted by Rhipicephalus sanguineus ticks. However, only a few clinical studies evaluating dogs infected with these organisms and anemia condition have been published. In this study, the potential tick-borne bacteria linked to anemia were investigated in eighty-one blood samples selected from anemic dogs using a broad range nested-PCR of the 16S rRNA gene. Positive results were shown in 12/81 blood specimens (14.81%). Nucleotide sequences from the PCR products were analyzed using BLAST and resulted in identification of Ehrlichia canis (8), Candidatus Mycoplasma haematoparvum (1) and Anaplasma platys (3). Two other PCR assays were used to detect and identify the positive results of these pathogens including a specific PCR for Ehrlichia canis (gp36) and a specific nested-PCR for hemoplasma species (16S rRNA) and the phylogenetic analyses of E. canis and canine hemoplasmas were performed using these two loci. These specific PCRs revealed co-infection of E. canis and Mycoplasma haemocanis in two cases. These two male dogs had presented with jaundice, severe hemolytic anemia, severe thrombocytopenia, leukocytosis, mild azotemia and hepatitis. Ehrlichia canis was detected in a significantly greater number of severe anemia cases (PCV<15%) than moderate or mild anemia cases (PCV 16-29%) (P<0.05) and these severe anemia cases were 7-fold more at risk of having E. canis infections (odds ratio: 7.11, p=0.020). However, no statistical differences were detected between E. canis detection and degrees of thrombocytopenia or leukopenia. From the results of this study, we conclude that the severity of anemia is associated with E. canis infections rather than the severity of thrombocytopenia.

Analgesic efficacy of oral firocoxib in ovariohysterectomized cats

The postoperative analgesic effects of firocoxib in ovariohysterectomized cats were observed. Twenty-four cats were divided into 3 groups: control (no medicine), firocoxib-1 (1 mg/kg/day) and firocoxib-3 (3 mg/kg/day). Colorado pain scale scores (CPSS), composite pain scores (CPS), and buccal mucosal bleeding times (BMBT) were recorded in blinded fashion before induction and 2, 5, 8, 24, 30, and 48 h post-operation. The average CPSS (mean ± SEM) over 2 to 48 h post-operation in firocoxib-3 (0.4 ± 0.1) was significantly lower than that of the control (0.7 ± 0.2; p = 0.004), but that of firocoxib-1 (0.5 ± 0.2) was not different from that of the control (p = 0.40). The mean CPS of firocoxib-3 was significantly lower than that of the control at 24 h post-operation (p = 0.04); nonetheless, there was no significant difference in mean CPS between firocoxib-1 and control groups at all intervals. BMBT and body temperature were within normal limits in all groups. However, reversible azotemia was identified in two firocoxib-3 cats at 72 h post-operation. One firocoxib-3 cat vomited once at 48 h post-operation. In conclusion, firocoxib-3 is helpful for postoperative pain control in cats; however, gastrointestinal irritation and renal function side effects may occur.

Development and analytic validation of an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-bromotyrosine in canine serum

BACKGROUND The activation of eosinophils causes the release of eosinophil peroxidase and subsequent production of 3-bromotyrosine (3-BrY), a stable byproduct. In people, 3-BrY is used as a biomarker for eosinophil activation. The method for measuring 3-BrY concentrations in biologic samples from dogs has not previously been described. OBJECTIVES The objective of this study was to develop and analytically validate an electron ionization gas chromatography/mass spectrometry (EI-GC/MS) method for the measurement of 3-BrY in canine serum samples. METHODS Pooled canine serum samples were utilized to validate the assay. Serum samples from healthy control dogs (n = 41) were used to evaluate 3-BrY concentrations and establish a reference interval. RESULTS The analytic validation revealed that the limit of blank and limit of detection were 0.33 and 0.63 μmol/L, respectively. The coefficients of variation for precision and reproducibility for 3-BrY were < 13.9% and < 11.0%, respectively. The means ± SD of observed-to-expected ratios for linearity and accuracy were 109.6 ± 17.2% and 98.7 ± 11.3%, respectively. The reference interval was determined as ≤ 1.12 μmol/L (median [range]: ≤ 0.63 μmol/L [≤ 0.63-1.13]). CONCLUSIONS The EI-GC/MS assay described here for the measurement of 3-BrY in canine serum samples was precise, reproducible, linear, and accurate. Further studies are underway to determine the diagnostic utilities in canine patients with eosinophilic diseases.

Comparison of post-operative analgesic efficacy of tolfenamic acid and robenacoxib in ovariohysterectomized cats

The objective of this study was to evaluate the efficacy of a non-selective COX inhibitor (tolfenamic acid) and a selective COX-2 inhibitor (robenacoxib) for post-operative pain control in cats. Thirty cats undergoing ovariohysterectomy were randomly divided into three groups: the control (placebo) group, the tolfenamic acid (4 mg/kg/day) group, and the robenacoxib (1 mg/kg/day) group. Non-steroidal anti-inflammatory drugs (NSAIDs) were administered orally 2 hr before anesthesia induction and 24 and 48 hr post-operation. Buccal mucosal bleeding times (BMBTs) were assessed prior to anesthesia induction. Colorado pain scores and composite pain scores were evaluated in a blinded fashion before induction and 2, 8, 24, 30 and 48 hr post-operation. The Colorado pain scores of cats receiving robenacoxib were significantly lower than those of cats in the control group at 30 (P=0.0126) and 48 (P=0.0439) hr post-operation. The composite pain scores of cats from the robenacoxib group were lower than those of cats in the control group at 30 (P=0.0299) and 48 (P=0.0103) hr post-operation. The Colorado pain scores of cats receiving tolfenamic acid were significantly lower than those of cats in the control group at 30 hr (P=0.0186) post-operation. The composite pain scores in cats in the tolfenamic acid group were lower than the scores of cats in the control group at 24 (P=0.0403) and 48 (P=0.0413) hr post-operation. BMBTs remained within normal limits in all groups. Both tolfenamic acid and robenacoxib are useful for post-operative pain control in cats.