Deletion of CD73 in mice leads to aortic valve dysfunction.

Aortic stenosis is known to involve inflammation and thrombosis. Changes in activity of extracellular enzyme - ecto-5'-nucleotidase (referred also as CD73) can alter inflammatory and thrombotic responses. This study aimed to evaluate the effect of CD73 deletion in mice on development of aortic valve dysfunction and to compare it to the effect of high-fat diet. Four groups of mice (normal-diet Wild Type (WT), high-fat diet WT, normal diet CD73-/-, high-fat diet CD73-/-) were maintained for 15weeks followed by echocardiographic analysis of aortic valve function, measurement of aortic surface activities of nucleotide catabolism enzymes as well as alkaline phosphatase activity, mineral composition and histology of aortic valve leaflets. CD73-/- knock out led to an increase in peak aortic flow (1.06±0.26m/s) compared to WT (0.79±0.26m/s) indicating obstruction. Highest values of peak aortic flow (1.26±0.31m/s) were observed in high-fat diet CD73-/- mice. Histological analysis showed morphological changes in CD73-/- including thickening and accumulation of dark deposits, proved to be melanin. Concentrations of Ca2+, Mg2+ and PO43- in valve leaflets were elevated in CD73-/- mice. Alkaline phosphatase (ALP) activity was enhanced after ATP treatment and reduced after adenosine treatment in aortas incubated in osteogenic medium. AMP hydrolysis in CD73-/- was below 10% of WT. Activity of ecto-adenosine deaminase (eADA), responsible for adenosine deamination, in the CD73-/- was 40% lower when compared to WT. Deletion of CD73 in mice leads to aortic valve dysfunction similar to that induced by high-fat diet suggesting important role of this surface protein in maintaining heart valve integrity.

Long-term maintenance therapy for post-cardiac transplant monoclonal lymphoproliferative disorder: caveat mammalian target of rapamycin.

A 53-year-old Caucasian male suffering from idiopathic dilated cardiomyopathy underwent cardiac transplantation. Fifty-seven days following transplant, he developed posttransplant lymphoproliferative disorder (PTLD), which was Epstein-Barr virus positive. The initial episode of PTLD was treated with a dose reduction in cyclosporine (CsA) and a 4-week course of rituximab. Subsequent biopsies showed resolution of PTLD. One year posttreatment, his evaluation revealed severe cardiac allograft vasculopathy (CAV). The patient was switched to sirolimus-based immunosuppression regimen with gradual up-titration of sirolimus in combination with complete withdrawal of previously administered Calcineurin-based immunosuppression approach. The switchover was carried out over a 6-week period. In the following 3 years, there was CAV regression as well as PTLD remission, without any significant episode of rejection. Despite frequent relapses with this form of PTLD, the patient remains in remission, 8 years posttransplantation. In summary, sirolimus has been demonstrated to attenuate the progression of CAV, and this case report illustrates that regression of CAV is possible. In addition to preventing rejection, mammalian target of rapamycin inhibitors directly suppress signaling pathways leading to PTLD and may be effective monotherapy for preventing rejection and suppressing PTLD.

Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation.

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.

Purine metabolism in pigs and humans and its implications for xenotransplantation.

We compared concentrations of nucleotide substrates and activities of enzymes of nucleotide metabolism in pig and human blood, heart, and kidney. The most important difference was lower ecto-5-nucleotidase (ESN) activity in both pig hearts and kidney. Furthermore, higher hypoxanthine, inosine, adenine, and uracil, but lower uridine and uric acid concentrations were observed in pig blood as compared to human. A twofold increase in UTP concentration has been observed in pig hearts following 4 h perfusion with human blood. Purine metabolism is an important target for genetic and pharmacological manipulation during xenotransplantations.

Exposure to human blood inactivates swine endothelial ecto-5'-nucleotidase.

Ecto-5'-nucleotidase (E5'N) is an extracellular enzyme forming anti-inflammatory and immunosuppressive adenosine. We evaluated whether confrontation of pig heart and endothelial cells with human blood changes the activity of E5'N. Pig hearts were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were incubated in vitro with human plasma for 3 h. Ex vivo perfusion of pig heart with fresh human blood resulted in a decrease in E5'N activity to 62% and 61% of initial in wild-type and transgenic pig hearts, respectively. PAEC activity of E5'N decreased to 71% and 50% of initial after 3 h exposure to heat-inactivated and active complement human plasma, respectively, while it remained constant in controls. Pig heart activity of E5'N decreased following exposure to human blood, which may affect adenosine production and exacerbate hyperacute and vascular rejection.

Cellular physiology of mismatch repair.

The DNA mismatch repair system maintains genomic stability by correcting DNA sequence errors generated during DNA replication, during genetic exchanges between chromosomes i.e., recombination, and by correcting DNA lesions caused by mutagenic agents such as cis-platinum. Post-synthesis mismatch repair improves almost 1000-fold the fidelity of DNA replication; however, the functions of mismatch repair proteins extend well beyond DNA repair. Recent studies suggest that mismatch repair is part of the machinery that couples DNA damage and repair to cell cycle regulation and apoptosis. These studies indicate that tolerance to certain DNA lesions (such as methylation and cis-platinum adducts) is associated with inefficient activation of cell cycle checkpoints and inefficient activation of apoptosis in mismatch repair deficient cells. Hence, mismatch repair proteins regulate the survival threshold to DNA damage, and this function provides a novel platform for understanding the role of mismatch repair in B cells, in tumor formation, as well as in resistance to chemotherapy. In this communication, we review how mismatch repair may contribute to the physiology of cells and may be regulated by the intracellular trafficking of mismatch repair proteins.