Michael Angelo

S-nitrosohemoglobin deficiency: A mechanism for loss of physiological activity in banked blood

RBCs distribute oxygen to tissues, but, paradoxically, blood transfusion does not always improve oxygen delivery and is associated with ischemic events. We hypothesized that storage of blood would result in loss of NO bioactivity, impairing RBC vasodilation and thus compromising blood flow, and that repleting NO bioactivity would restore RBC function. We report that S-nitrosohemoglobin (SNO-Hb) concentrations declined rapidly after storage of fresh venous blood and that hypoxic vasodilation by banked RBCs correlated strongly with the amounts of SNO-Hb (r2 = 0.90; P < 0.0005). Renitrosylation of banked blood during storage increased the SNO-Hb content and restored its vasodilatory activity. In addition, canine coronary blood flow was greater during infusion of renitrosylated RBCs than during infusion of S-nitrosothiol-depleted RBCs, and this difference in coronary flow was accentuated by hypoxemia (P < 0.001). Our findings indicate that NO bioactivity is depleted in banked blood, impairing the vasodilatory response to hypoxia, and they suggest that SNO-Hb repletion may improve transfusion efficacy.

Assessment of nitric oxide signals by triiodide chemiluminescence

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures.

Myocyte Specific Overexpression of Myoglobin Impairs Angiogenesis After Hind-Limb Ischemia

Objective—In preclinical models of peripheral arterial disease the angiogenic response is typically robust, though it can be impaired in conditions such as hypercholesterolemia and diabetes where the endothelium is dysfunctional. Myoglobin (Mb) is expressed exclusively in striated muscle cells. We hypothesized that myocyte specific overexpression of myoglobin attenuates ischemia-induced angiogenesis even in the presence of normal endothelium. Methods and Results—Mb overexpressing transgenic (MbTg, n=59) and wild-type (WT, n=56) C57Bl/6 mice underwent unilateral femoral artery ligation/excision. Perfusion recovery was monitored using Laser Doppler. Ischemia-induced changes in muscle were assessed by protein and immunohistochemistry assays. Nitrite/nitrate and protein-bound NO, and vasoreactivity was measured. Vasoreactivity was similar between MbTg and WT. In ischemic muscle, at d14 postligation, MbTg increased VEGF-A, and activated eNOS the same as WT mice but nitrate/nitrite were reduced whereas protein-bound NO was higher. MbTg had attenuated perfusion recovery at d21 (0.37±0.03 versus 0.47±0.02, P<0.05), d28 (0.40±0.03 versus 0.50±0.04, P<0.05), greater limb necrosis (65.2% versus 15%, P<0.001), a lower capillary density, and greater apoptosis versus WT. Conclusion—Increased Mb expression in myocytes attenuates angiogenesis after hind-limb ischemia by binding NO and reducing its bioavailability. Myoglobin can modulate the angiogenic response to ischemia even in the setting of normal endothelium.

InAs(100) Surfaces Cleaning by an As-Free Low-Temperature 100 ° C Treatment

Oxide removal from InAs(100) surfaces was achieved using a combination of HF:methanol wet etching followed by atomic hydrogen treatment at a temperature as low as 100°C without any stabilizing As flux. The process was monitored in real-time exploiting spectroscopic ellipsometry. Following this treatment, the surface morphology was found to be very smooth at the nanometer scale, with a reduced effective oxide thickness and no appreciable levels of elemental In and As. Furthermore, we demonstrate stability of such surfaces against oxide reformation.