Sildenafil as a Rescue Agent Following Intestinal Ischemia and Reperfusion Injury.

BACKGROUND: Acute mesenteric ischemia carries a significant morbidity. Measures to improve blood flow parameters to the intestine may ameliorate the disease. Sildenafil, a phosphodiesterase 5 inhibitor, increases cyclic guanosine monophosphate and has been shown to prevent the effects of ischemia when given before injury. However, its effects as a rescue agent have not been established. We therefore hypothesized that sildenafil, when given as a rescue agent for intestinal ischemia, would improve mesenteric perfusion, limit intestinal epithelial injury, and decrease intestinal leukocyte chemoattractants. METHODS: Eight to 12 wk-old-male C57BL/6J mice underwent laparotomy and temporary occlusion of the superior mesenteric artery for 60 min. Following ischemia, reperfusion was permitted, and before closing the abdomen, sildenafil was injected intraperitoneally in a variety of concentrations. After 24 h, reperfusion was reassessed. Animals were euthanized and intestines evaluated for histologic injury and leukocyte chemoattractants. RESULTS: Postischemic administration of sildenafil did not improve mesenteric perfusion following intestinal ischemia and reperfusion injury. However, sildenafil did improve histologic injury scores in dose ranges of 0.01 to 10 mg/kg. No difference was noted in histological injury with 100 mg/kg dose, and all members of the 1000 mg/kg group died within 24 h of injury. Epithelial protection was not facilitated by the leukocyte chemoattractants Regulated on Activation, Normal T Cell Expressed, and Secreted, macrophage inflammatory protein 1 alpha, monocyte chemoattractant protein, neutrophil activating protein, or granulocyte colony stimulating factor. CONCLUSIONS: Administration of sildenafil following intestinal ischemia may limit intestinal mucosal injury but does not appear to alter mesenteric perfusion or leukocyte chemoattractant influx. TYPE: Basic science. LEVEL OF EVIDENCE: N/A.

Mycophenolic acid induces senescence of vascular precursor cells.

OBJECTIVE: Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function. METHODS: We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects. RESULTS: Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated ß-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA. CONCLUSION: Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells in vitro, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies in vivo to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.

Functional Differences Between Placental Micro- and Macrovascular Endothelial Colony-Forming Cells.

Alterations in the development of the placental vasculature can lead to pregnancy complications, such as preeclampsia. Currently, the cause of preeclampsia is unknown, and there are no specific prevention or treatment strategies. Further insight into the placental vasculature may aid in identifying causal factors. Endothelial colony-forming cells (ECFCs) are a subset of endothelial progenitor cells capable of self-renewal and de novo vessel formation in vitro. We hypothesized that ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Human placentas were collected from term pregnancies delivered by cesarean section (n = 16). Placental micro- and macrovasculature was collected from the maternal and fetal side of the placenta, respectively, and ECFCs were isolated and characterized. ECFCs were CD31(+), CD105(+), CD144(+), CD146(+), CD14(-), and CD45(-), took up 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein, and bound Ulex europaeus agglutinin 1. In vitro, macrovascular ECFCs had a greater potential to generate high-proliferative colonies and formed more complex capillary-like networks on Matrigel compared with microvascular ECFCs. In contrast, in vivo assessment demonstrated that microvascular ECFCs had a greater potential to form vessels. Macrovascular ECFCs were of fetal origin, whereas microvascular ECFCs were of maternal origin. ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Although macrovascular ECFCs demonstrated greater vessel and colony-forming potency in vitro, this did not translate in vivo, where microvascular ECFCs exhibited a greater vessel-forming ability. These important findings contribute to the current understanding of normal placental vascular development and may aid in identifying factors involved in preeclampsia and other pregnancy complications.

Changes in the frequency and in vivo vessel-forming ability of rhesus monkey circulating endothelial colony-forming cells across the lifespan (birth to aged).

INTRODUCTION: We have identified a novel hierarchy of human endothelial colony-forming cells (ECFCs) that are functionally defined by their proliferative and clonogenic potential and in vivo vessel-forming ability. The rhesus monkey provides an excellent model in which to examine the changes in circulating concentrations and functions of ECFCs since this nonhuman primate possesses a long lifespan and has been used extensively to model age-related processes that occur in humans. RESULTS: Endothelial cells (ECs) derived from rhesus monkey ECFCs share a cell-surface phenotype similar to human cord blood ECFCs, rapidly form capillary-like structures in vitro, and form endothelial-lined vessels in vivo upon implantation in immunodeficient mice in an age-dependent manner. Of interest, although ECFCs from the oldest monkeys formed capillary-like structures in vitro, the cells failed to form inosculating vessels when implanted in vivo and displayed a deficiency in cytoplasmic vacuolation in vitro; a critical first step in vasculogenesis. DISCUSSION: Utilizing previously established clonogenic assays for defining different subpopulations of human ECFCs, we have shown that a hierarchy of ECFCs, identical to human cells, can be isolated from the peripheral blood of rhesus monkeys, and that the frequency of the circulating cells varies with age. These studies establish the rhesus monkey as an important preclinical model for evaluating the role and function of circulating ECFCs in vascular homeostasis and aging. METHODS: Peripheral blood samples were collected from 40 healthy rhesus monkeys from birth to 24 years of age for ECFC analysis including immunophenotyping, clonogenic assays, and in vivo vessel formation.

Shp-2 heterozygous hematopoietic stem cells have deficient repopulating ability due to diminished self-renewal.

OBJECTIVE: Improved understanding of hematopoietic stem cell (HSC) differentiation, proliferation, and self-renewal is sought to develop improved stem cell-based therapies as well as to define novel therapies for stem cell-based diseases such as leukemia. Shp-2 is a widely expressed nonreceptor protein tyrosine phosphatase that participates early in hematopoietic development. The following study was performed to examine the role of Shp-2 in HSC function. METHODS: Bone marrow low-density mononuclear cells were isolated from WT and Shp-2(+/-) littermate controls and utilized in competitive repopulation studies, homing analysis, cell-cycle analysis, and serial transplantation studies. RESULTS: Haploinsufficiency of Shp-2 causes a threefold reduction in HSC repopulating units following transplantation into lethally irradiated recipients. Homing of Shp-2(+/-) and WT cells to the bone marrow and spleen compartments was equal. Cell-cycle analysis studies revealed that the Shp-2(+/-) lin(-)Sca-1(+)c-kit(+) cells are less quiescent than WT cells, providing a potential etiology for the observed reduced engraftment of the Shp-2(+/-) cells. Consistently, in serial transplantation studies, we observed a significant reduction of Shp-2(+/-) self-renewal compared to that of WT cells. CONCLUSION: These data demonstrate that Shp-2 is required for the physiologic homeostasis of the HSC compartment and potentially provide insight into how oncogenic Shp-2 may contribute to the pathogenesis of myeloproliferative disorders and leukemias.