Nucleotide Catabolism on the Surface of Aortic Valve Xenografts; Effects of Different Decellularization Strategies.

Extracellular nucleotide metabolism controls thrombosis and inflammation and may affect degeneration and calcification of aortic valve prostheses. We evaluated the effect of different decellularization strategies on enzyme activities involved in extracellular nucleotide metabolism. Porcine valves were tested intact or decellularized either by detergent treatment or hypotonic lysis and nuclease digestion. The rates of ATP hydrolysis, AMP hydrolysis, and adenosine deamination were estimated by incubation of aorta or valve leaflet sections with substrates followed by HPLC analysis. We demonstrated relatively high activities of ecto-enzymes on porcine valve as compared to the aortic wall. Hypotonic lysis/nuclease digestion preserved >80 % of ATP and AMP hydrolytic activity but reduced adenosine deamination to <10 %. Detergent decellularization completely removed (<5 %) all these activities. These results demonstrate high intensity of extracellular nucleotide metabolism on valve surface and indicate that various valve decellularization techniques differently affect ecto-enzyme activities that could be important in the development of improved valve prostheses.

Quantitative assessment of initial retention of bone marrow mononuclear cells injected into the coronary arteries.

BACKGROUND: Intracoronary injection of bone marrow mononuclear cells (BMMNC) is a common clinical protocol of cell transplantation for heart disease, but poor engraftment of donor cells in the heart, which will limit its therapeutic efficacy, is a major issue. Initial "retention" (endothelial adherence and/or extravasation) of BMMNC immediately after intracoronary injection is a key step toward successful engraftment; however, this event has not been fully characterized. The aim of this study is to quantitatively clarify the frequency of "retention" of BMMNC after intracoronary injection, determine the impact of prior induction of ischemia-reperfusion injury on "retention" efficiency, and elucidate the underlying mechanisms focusing on adhesion molecule-mediated cell-cell interactions. METHODS: One million BMMNC collected from green fluorescent protein (GFP)-transgenic mice were injected into the coronary arteries of syngeneic wild-type mouse hearts under Langendorff perfusion. Retention efficiency was quantitatively estimated from the GFP-positive cell number flushed out into the coronary effluent. RESULTS: Whereas only 13.3 ± 1.2% of injected BMMNC were retained into normal hearts, prior induction of 30-minute ischemia and 30-minute reperfusion increased the retention efficiency to 36.5 ± 1.6% (p < 0.05, n = 8). Immunoconfocal observation further confirmed this enhanced retention after ischemia-reperfusion. Noticeably, the enhanced retention efficiency after ischemia-reperfusion treatment was diminished by administration of anti-P-selectin antibody (8.3 ± 0.8%, p < 0.05), but was not affected by inhibiting intercellular adhesion molecule-1 (39.6 ± 3.3%) or vascular cell adhesion molecule-1 (43.9 ± 2.9%). CONCLUSIONS: Retention efficiency of intracoronary-injected BMMNC was poor in a model of isolated, crystalloid-perfused murine hearts. An antecedent period of global ischemia-reperfusion increased the retention via P-selectin-dependent BMMNC-endothelial interaction.

Erythrocyte-dependent regulation of human skeletal muscle blood flow: role of varied oxyhemoglobin and exercise on nitrite, S-nitrosohemoglobin, and ATP.

The erythrocyte is proposed to play a key role in the control of local tissue perfusion via three O(2)-dependent signaling mechanisms: 1) reduction of circulating nitrite to vasoactive NO, 2) S-nitrosohemoglobin (SNO-Hb)-dependent vasodilatation, and 3) release of the vasodilator and sympatholytic ATP; however, their relative roles in vivo remain unclear. Here we evaluated each mechanism to gain insight into their roles in the regulation of human skeletal muscle blood flow during hypoxia and hyperoxia at rest and during exercise. Arterial and femoral venous hemoglobin O(2) saturation (O(2)Hb), plasma and erythrocyte NO and ATP metabolites, and leg and systemic hemodynamics were measured in 10 healthy males exposed to graded hypoxia, normoxia, and graded hyperoxia both at rest and during submaximal one-legged knee-extensor exercise. At rest, leg blood flow and NO and ATP metabolites in plasma and erythrocytes remained unchanged despite large alterations in O(2)Hb. During exercise, however, leg and systemic perfusion and vascular conductance increased in direct proportion to decreases in arterial and venous O(2)Hb (r(2) = 0.86-0.98; P = 0.01), decreases in venous plasma nitrite (r(2) = 0.93; P < 0.01), increases in venous erythrocyte nitroso species (r(2) = 0.74; P < 0.05), and to a lesser extent increases in erythrocyte SNO (r(2) = 0.59; P = 0.07). No relationship was observed with plasma ATP (r(2) = 0.01; P = 0.99) or its degradation compounds. These in vivo data indicate that, during low-intensity exercise and hypoxic stress, but not hypoxic stress alone, plasma nitrite consumption and formation of erythrocyte nitroso species are associated with limb vasodilatation and increased blood flow in the human skeletal muscle vasculature.

Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation.

Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors.Ecto-5'-nucleotidase (E5'N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.

Decreased cardiac activity of AMP deaminase in subjects with the AMPD1 mutation--a potential mechanism of protection in heart failure.

OBJECTIVES: Possession of the C34T (Glu12Stop) nonsense mutation in the AMP-deaminase 1 (AMPD1) gene has been shown to be associated with improved prognosis in heart failure and ischemic heart disease. The most likely event leading to these clinical effects is a reduced capacity of the AMP deamination pathway and increased production of cardio-protective adenosine. However, since AMPD1 is predominantly expressed in skeletal muscle, the protective effects could be related not only to local cardiac changes, but also to a systemic mechanism. In the present study we evaluated the effect of the C34T mutation on cardiac AMP-deaminase activity and on the systemic changes in adenosine production. METHODS: The presence of the C34T mutation was assayed by single-stranded conformational polymorphism (SSCP). Analysis of the AMPD1 genotype and measurement of enzyme activities was performed on 27 patients with heart failure (HF). In addition, blood adenosine concentration was measured by liquid chromatography/mass spectrometry (LC/MS) in 21 healthy subjects with established AMPD1 genotype at rest and following exhaustive exercise. RESULTS: Cardiac AMP-deaminase activity in heterozygotes (C/T) was 0.59+/-0.02 nmol/min/g wet wt-about half of the activity found in normal wild-type (C/C) individuals (1.06+/-0.09 nmol/min/g wet wt, P=0.003). There were no significant differences in the activities of any other enzymes between subjects with the C/T or C/C genotype. Resting venous blood adenosine concentration was similar in subjects with C/C, C/T and homozygous for the mutated allele (T/T) genotype. Following exercise, a significant increase in adenosine was observed in T/T subjects (by 0.013+/-0.009 micromol/l, P=0.035) but not in C/C (0.003+/-0.009 micromol/l) or C/T (-0.002+/-0.011 micromol/l). CONCLUSIONS: Our findings indicate that the C34T mutation of AMPD1 leads to a decrease in cardiac enzyme activity of AMP-deaminase without changes in any other adenosine-regulating enzymes, highlighting the importance of local cardiac metabolic changes. Systemic (blood) changes in adenosine concentration were apparent only in homozygous subjects and therefore may play a relatively small part in cardio-protection.