Arvind Koshal

Endothelium-Derived Hyperpolarizing Factor in Human Internal Mammary Artery Is 11,12-Epoxyeicosatrienoic Acid and Causes Relaxation by Activating Smooth Muscle BKCa Channels

Background—Left internal mammary arteries (LIMAs) synthesize endothelium-derived hyperpolarizing factor (EDHF), a short-lived K+ channel activator that persists after inhibition of nitric oxide (NO) and prostaglandin synthesis. EDHF hyperpolarizes and relaxes smooth muscle cells (SMCs). The identity of EDHF in humans is unknown. We hypothesized that EDHF (1) is 11,12-epoxyeicosatrienoic acid (11,12-EET); (2) is generated by cytochrome P450-2C, CYP450-2C; and (3) causes relaxation by opening SMC large-conductance Ca2+-activated K+ channels (BKCa). Methods and Results—The identity of EDHF and its mechanism of action were assessed in 120 distal human LIMAs and 20 saphenous veins (SVs) obtained during CABG. The predominant EET synthesized by LIMAs is 11,12-EET. Relaxations to exogenous 11,12-EET and endogenous EDHF are of similar magnitudes. Inhibition of EET synthesis by chemically distinct CYP450 inhibitors (17-octadecynoic acid, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide), or a selective EET antagonist (4,15-epoxyeicosa-5(Z)-enoic acid) impairs EDHF relaxation. 11,12-EET activates a BKCa current and hyperpolarizes LIMA SMCs. Inhibitors of BKCa but not inward-rectifier or small-conductance KCa channels abolish relaxation to endogenous EDHF and exogenous 11,12-EET. BKCa and CYP450-2C mRNA and proteins are more abundant in LIMAs than in SVs, perhaps explaining the lack of EDHF activity of the SV. Laser capture microdissection and quantitative RT-PCR demonstrate that BKCa channels are primarily in vascular SMCs, whereas the CYP450-2C enzyme is present in both the endothelium and SMCs. Conclusions—In human LIMAs, EDHF is 11,12-EET produced by an EDHF synthase CYP450-2C and accounting for ≈40% of net endothelial relaxation. 11,12-EET causes relaxation by activating SMC BKCa channels.

In Vivo Gene Transfer of the O2-Sensitive Potassium Channel Kv1.5 Reduces Pulmonary Hypertension and Restores Hypoxic Pulmonary Vasoconstriction in Chronically Hypoxic Rats

Background—Alveolar hypoxia acutely elicits pulmonary vasoconstriction (HPV). Chronic hypoxia (CH), despite attenuating HPV, causes pulmonary hypertension (CH-PHT). HPV results, in part, from inhibition of O2-sensitive, voltage-gated potassium channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). CH decreases Kv channel current/expression and depolarizes and causes Ca2+ overload in PASMCs. We hypothesize that Kv gene transfer would normalize the pulmonary circulation (restore HPV and reduce CH-PHT), despite ongoing hypoxia. Methods and Results—Adult male Sprague-Dawley rats were exposed to normoxia or CH for 3 to 4 weeks and then nebulized orotracheally with saline or adenovirus (Ad5) carrying genes for the reporter, green fluorescent protein reporter±human Kv1.5 (cloned from normal PA). HPV was assessed in isolated lungs. Hemodynamics, including Fick and thermodilution cardiac output, were measured in vivo 3 and 14 days after gene therapy by use of micromanometer-tipped catheters. Transgene expression, measured by quantitative RT-PCR, was confined to the lung, persisted for 2 to 3 weeks, and did not alter endogenous Kv1.5 levels. Ad5-Kv1.5 caused no mortality or morbidity, except for sporadic, mild elevation of liver transaminases. Ad5-Kv1.5 restored the O2-sensitive K+ current of PASMCs, normalized HPV, and reduced pulmonary vascular resistance. Pulmonary vascular resistance decreased at day 2 because of increased cardiac output, and remained reduced at day 14, at which time there was concomitant regression of right ventricular hypertrophy and PA medial hypertrophy. Conclusions—Kv1.5 is an important O2-sensitive channel and potential therapeutic target in PHT. Kv1.5 gene therapy restores HPV and improves PHT. This is, to the best of our knowledge, the first example of K+ channel gene therapy for a vascular disease.

Glutaraldehyde-Fixed Bioprosthetic Heart Valve Conduits Calcify and Fail From Xenograft Rejection

Background— Glutaraldehyde fixation (G-F) decreases but likely does not eliminate the antigenicity of bioprosthetic heart valves. Rejection (with secondary dystrophic calcification) may be why G-F xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Therefore, we sought to determine whether rejection of G-F xenograft occurs and to correlate this with graft calcification. Methods and Results— Ascending aortas/valves (from rats [syngeneic] or guinea pigs [xenogeneic]) were transplanted (fresh or after 48 hour of G-F) into the infrarenal aortas of young rat recipients for 20 days. A xenogeneic group was also treated with steroids until graft harvest. The valves and media/adventitia were scored blindly for inflammation (0 to 4). Percent graft infiltration by T cells/macrophages was determined (immunohistochemistry), and rat IgG ELISAs were performed. There was >3 times more valve inflammation, >10 times more valve T-cell/macrophage infiltrate, and >3 times antibody rise in the G-F xenogeneic groups compared with the fresh syngeneic or the G-F syngeneic groups (P<0.05). There was >2 times more adventitial inflammation and T-cell/macrophage infiltrate in the xenogeneic groups (P<0.05). Steroid treatment decreased inflammation and antibody rise in the xenogeneic groups (P<0.05). Correlation analysis revealed media/adventitia inflammation (P=0.02) and percent macrophage (P=0.01) infiltration to be predictors of calcification. Conclusions— G-F xenografts have cellular/humoral rejection and calcify secondarily.

Magnesium sulfate prophylaxis after cardiac operations

One hundred patients undergoing elective cardiac operations were randomized into placebo (n = 54) and magnesium (n = 46) groups. The magnesium group received six doses of 2.4 g (19.2 mEq) magnesium sulfate intravenously in the first 24 hours after the cardiac operation. The magnesium group had higher serum magnesium concentrations postoperatively (1.09 +/- 0.20 versus 0.75 +/- 0.13 mmol/L; p < 0.0001), postoperative day 1 (1.49 +/- 0.34 versus 0.70 +/- 0.12 mmol/L; p < 0.0001) and postoperative day 2 (0.96 +/- 0.19 versus 0.76 +/- 0.07 mmol/L; p < 0.0001). Patients in the magnesium group had a lower incidence of ventricular tachyarrythmias (VTs) (17.3% versus 51.9%; p = 0.0006), less need for treatment (6.5% versus 20.3%; p < 0.0001), fewer VT episodes/patient (0.3 +/- 0.8 versus 1.39 +/- 1.9; p < 0.0001), and a reduction in the severity of VTs as measured by the modified Lown grade (p = 0.0002). No differences were demonstrated with respect to supraventricular tachyarrythmias. The magnesium group had reduced absolute creatine kinase-MB levels (5.3 +/- 4.2 versus 28.4 +/- 28 IU/L; p = 0.001) as well as creatine kinase-MB fraction (0.01 +/- 0.02 versus 0.05 +/- 0.04; p = 0.001) on postoperative day 1. Serum magnesium concentrations were lower during VTs than during periods of sinus rhythm (0.75 +/- 0.75 versus 1.02 +/- 0.35 mmol/L; p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Natural human antibody-mediated destruction of porcine neonatal islet cell grafts

Abstract: Porcine pancreata may be considered a potential source of islets for transplantation into diabetic recipients; however, whether porcine islet grafts will be susceptible to damage by natural antibody‐mediated hyperacute rejection remains unknown. In this study, we performed Western blots to determine whether membrane proteins present on porcine neonatal islet cells (NIC) are recognized by xenoreactive antibodies present in human sera. Western blots of freshly isolated porcine NICs with AB sera detected the presence of 14 antigens (MW 24–164 kDa) and 4 antigens (MW 101–150 kDa) to which antiserum against human IgM and IgG bound, respectively. The most prominent antigens with IgM reactivity had MWs of 36, 63, and 120 kDa, whereas for IgG, the most intensely reactive antigen had a MW of 120 kDa. When membrane fractions prepared from purified porcine aortic endothelial cells and LLC‐PK1 cells were analyzed, the major antigens had molecular weights comparable to those seen for NICs. After culturing the NICs for 5 days, the number of detected xenoreactive antigens binding IgM or IgG decreased and the antigens present at 36, 63, and 120 kDa with IgM reactivity were shown to have a decreased intensity of binding. Incubation of cultured porcine NICs for 18 hr in the presence of human AB serum containing complement resulted in a 55% loss of cellular insulin content (P < 0.0001), a 45% reduction in recoverable DNA (P < 0.0001), and a marked reduction in insulin secretory response to an in vitro glucose challenge. Recovery and viability of porcine NICs was not affected when incubated with AB serum depleted of anti‐Gal antibodies with Synsorb 90. These results demonstrate that natural human antibodies of both IgM and IgG subtypes bind to antigens present on Department of Laboratory Medicine and complement reduces islet cell survival and functional viability. Adsorbing serum with the αGal(1–3)βGal(1–4)βGlc carbohydrate removes natural human antibody‐mediated destruction of porcine neonatal islet cell grafts.

Doxycycline reduces cardiac matrix metalloproteinase-2 activity but does not ameliorate myocardial dysfunction during reperfusion in coronary artery bypass patients undergoing cardiopulmonary bypass.

Objectives:Matrix metalloproteinase-2 proteolyzes intracellular proteins in the heart and induces acute myocardial contractile dysfunction in ischemia-reperfusion injury. Doxycycline, a matrix metalloproteinase inhibitor, prevented matrix metalloproteinase-2-induced troponin I cleavage in rat hearts and improved contractile function following ischemia-reperfusion. In patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass, increased atrial matrix metalloproteinase-2 activity was inversely correlated with cardiac mechanical function at 3 hours reperfusion. We performed a study in patients with coronary artery disease undergoing primary elective coronary artery bypass graft surgery with cardiopulmonary bypass to determine whether doxycycline reduces cardiac mechanical dysfunction, matrix metalloproteinase activity, and troponin I degradation after reperfusion. Design:Randomized, double-blinded, placebo-controlled study. Setting:University of Alberta Hospital. Patients:Forty-two patients with coronary artery disease undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. Interventions:Patients were randomized to receive either oral administration of 20 mg of doxycycline or matching placebo pill twice a day at least 2 days prior to surgery, on the day of surgery, and for the first 3 postoperative days. Measurements and Main Results:Left ventricular stroke work index was examined prior to cardiopulmonary bypass and at 24 hours reperfusion. Right atrial biopsies were collected before cardiopulmonary bypass and 10 minutes after aortic cross-clamp release to determine matrix metalloproteinase-2 activity and troponin I level. Blood was collected to determine matrix metalloproteinase activity and interleukin-6, C-reactive protein, and troponin I levels. Cardiac 72-kDa matrix metalloproteinase-2 activity was lower upon reperfusion in biopsies from the doxycycline group (p = 0.01), and the increase of matrix metalloproteinase-2 activity in the placebo group due to reperfusion did not appear in the doxycycline group (p = 0.05). Doxycycline, however, did not ameliorate cardiac mechanical dysfunction following reperfusion or the cardiopulmonary bypass-coronary artery bypass graft-induced increased plasma matrix metalloproteinase-9, interleukin-6, and C-reactive protein levels. Cardiopulmonary bypass-coronary artery bypass graft or doxycycline did not change tissue or plasma troponin I levels at 10 minutes reperfusion. Conclusions:Although doxycycline did not improve myocardial stunning following coronary artery bypass graft surgery with cardiopulmonary bypass, it reduced cardiac matrix metalloproteinase-2 activity in these patients. A larger trial and/or higher dose of doxycycline may yet be warranted.

Selective antegrade cerebral perfusion during aortic arch surgery confers survival and neuroprotective advantages

OBJECTIVE To assess the impact of using antegrade cerebral perfusion during aortic arch surgery on postoperative survival and neurologic outcomes. METHODS All operations were performed at the same hospital between January 2001 and January 2009. Patients undergoing aortic arch surgery using antegrade cerebral perfusion during deep hypothermia were compared with patients undergoing aortic arch surgery without antegrade cerebral perfusion during the same study period. Multivariable logistic regression and Cox proportional hazards model were used to identify predictors of postoperative cerebrovascular accidents and midterm survival, respectively. There were 46 patients in the antegrade cerebral perfusion group and 78 patients in the non-antegrade cerebral perfusion group. RESULTS There were no statistically significant differences in age, proportion of emergency operations, or proportion of type A aortic dissection between the 2 groups. There was a statistically significant and clinically important difference in the rates of postoperative cerebrovascular complications (2% antegrade cerebral perfusion vs 13% non-antegrade cerebral perfusion, P = .03), postoperative duration of mechanical ventilation (1.15 ± 0.19 days antegrade cerebral perfusion vs 2.13 ± 0.38 days non-antegrade cerebral perfusion, P = .02), and 3-year survival (93% antegrade cerebral perfusion vs 78% non-antegrade cerebral perfusion, P = .03). Antegrade cerebral perfusion was shown to be a significant predictor of reduced postoperative stroke rates and better survival at 3 years. CONCLUSIONS Antegrade cerebral perfusion was associated with improved survival and neurologic outcomes in patients undergoing aortic arch surgery, especially for cases requiring prolonged aortic arch repair periods.

Phosphorylcholine-coated circuits improve preservation of platelet count and reduce expression of proinflammatory cytokines in CABG: a prospective randomized trial.

Abstract  Background: The interaction of blood with foreign artificial surfaces during cardiopulmonary bypass (CPB) has been recognized as a major stimulus in evoking a systemic inflammatory and metabolic response. Phosphorylcholine (PC) is a new‐generation coating material designed to ameliorate biocompatibility and thereby to reduce the detrimental interactions of CPB. We studied the effects of PC‐coated perfusion circuits on platelet function and the humoral and cellular response to CPB. Methods: Thirty patients undergoing coronary artery bypass grafting were randomized to PC‐coated (PC group, n = 15) and noncoated (control group, n = 15) circuit groups. Clinical data, total blood loss, and pre‐ and postoperative platelet counts were recorded and IL‐6 and TNF‐α, CD41a, CD42b, and CD62p were measured at induction of anesthesia, after the initiation of CPB and at termination of CPB. Results: There was a significantly improved preservation of platelet count following CPB in the PC group (p = 0.028), which was sustained over a period of 72 hours. The use of PC‐coated circuits further resulted in a significant attenuation of TNF‐α and IL‐6 expression (p < 0.05 and p < 0.01); however, we were unable to detect any differences in clinical outcomes. Conclusions: Despite similar clinical outcome, the obvious reduction of cytokine expression and improved preservation of platelet count suggest superior biocompatibility of PC‐coated circuits.

Human ABO Blood Group Is Important in Survival and Function of Porcine Working Hearts

Pig organs express αGal antigen and thus are hyperacutely rejected if perfused by human blood. Human B/A antigens are similar to pig αGal antigen, suggesting that the corresponding antibodies may cross‐react. Our purpose was to determine if there is a human ABO blood‐group difference in porcine–human xenotransplantation. Plasma from six A, five B, seven AB, and six O individuals pooled by blood group were tested in an ex‐vivo porcine working heart model. Blood‐group A plasma‐perfused hearts survived 20 ± 14 min (n = 5), B 241 ± 9 min (n = 3), AB 151 ± 37 min (n = 5), and O 9 ± 1 min (n = 8). A and O were different (p < 0.001) from B and AB. Function was significantly better in group B. Edema accumulation and creatine kinase change was highest in A and O. All groups had comparable levels of anti‐αGal antibody, as well as comparable perfusion and operative conditions. Multivariate linear regression analysis showed the anti‐B antibody levels to be predictive of survival (p < 0.001). At higher plasma concentrations, hearts perfused with B plasma survived longer (p = 0.01) than AB (218 ± 45 min, n = 4 vs. 6 ± 0 min, n = 3). These results suggest a human ABO blood‐group difference in porcine‐to‐human xenotransplantation, which may be mediated by the anti‐A and anti‐B antibodies.

The management of non-traumatic cardiac arrest in the operating room with cardiopulmonary bypass

We present a case of a 29-year-old woman whom, while undergoing an elective gynecological procedure, acutely arrested. Closed chest cardiopulmonary compressions were not effective. Fortuitously, the cardiac surgical team was in an adjacent operating room, about to start an elective bypass case. After sternotomy, the patient was placed on cardiopulmonary bypass within 20 min of the arrest. The patient achieved return of spontaneous circulation and was ultimately discharged with only mild extremity weakness. The etiology of the arrest was never fully explained. Open chest massage and cardiopulmonary bypass should be considered early in the management of unexpected cardiac arrest, especially in the operating room where surgical expertise should be immediately available. Surgeons and anesthesiologists need to be aware of, and consider, the possibility of employing these techniques.