Oral tetrahydrobiopterin improves the beneficial effect of adenoviral-mediated eNOS gene transfer after induction of hindlimb ischemia.

We tested the hypothesis that oral supplementation with the endothelial nitric oxide synthase (eNOS) cofactor tetrahydrobiopterin (BH(4)) improved the therapeutic efficacy of eNOS gene transfer in the ischemic rat hindlimb. BH(4) or vehicle were begun 1 week before induction of hindlimb ischemia, whereas recombinant adenovirus containing bovine eNOS cDNA (AdeNOS) or vehicle [phosphate-buffered saline (PBS)] was infused intra-arterially into the ischemic hindlimb 10 days after induction of ischemia. Rats receiving co-treatment with dietary BH(4) and eNOS gene transfer (the [eNOS, +BH(4)] group) had greater eNOS expression, phospho-eNOS expression (Ser(1177)), Ca(2+)-dependent NOS activity, and nitrite + nitrate concentrations in the ischemic gastrocnemius than did rats receiving AdeNOS alone.

Oxidized low-density lipoprotein induces apoptosis in endothelial progenitor cells by inactivating the phosphoinositide 3-kinase/Akt pathway.

We tested the hypothesis that oxidized low-density lipoprotein (oxLDL)-induced inactivation of Akt within endothelial progenitor cells (EPCs) is mediated at the level of phosphoinositide 3-kinase (PI3K), specifically by nitrosylation of the p85 subunit of PI3K, and that this action is critical in provoking oxLDL-induced EPC apoptosis. Hypercholesterolemic ApoE null mice had a significant reduction of the phosphorylated Akt (p-Akt)/Akt ratio in EPCs, as well as a greater percentage of apoptosis in these cells than EPCs isolated from wild-type (WT) C57Bl/6 mice. EPCs were isolated from WT spleen and exposed to oxLDL in vitro.

Endothelial nitric oxide synthase affects both early and late collateral arterial adaptation and blood flow recovery after induction of hind limb ischemia in mice.

Objective: The goals of this study were to determine if endothelial nitric oxide synthase (eNOS) affects both early and late collateral arterial adaptation and blood flow recovery after severe limb ischemia in a mouse model and to determine if eNOS-derived NO is necessary for recruitment of chemokine (C-X-C motif) receptor 4 (CXCR4)(+) vascular endothelial growth factor receptor-1 (VEGFR1)(+) hemangiocytes to the site of ischemia.

Methods: Two studies were completed. In the first, hind limb ischemia was induced by unilateral femoral artery excision in three groups: C57Bl6 (wild-type), eNOS(-/-), and C57Bl/6 mice treated with N(G)-nitro-L-arginine methyl ester (L-NAME) from 1 day before excision through day 3 after excision (early L-NAME group).

Recovery from hind limb ischemia is less effective in type 2 than in type 1 diabetic mice: roles of endothelial nitric oxide synthase and endothelial progenitor cells.

Objective: We sought to directly compare the effects of type 1 and type 2 diabetes on postischemic neovascularization and evaluate the mechanisms underlying differences between these groups. We tested the hypothesis that type 2 diabetic mice have a greater reduction in endothelial nitric oxide synthase (eNOS) expression, a greater increase in oxidative stress, and reduced arteriogenesis and angiogenesis, resulting in less complete blood flow recovery than type 1 diabetic mice after induction of hind limb ischemia.

Methods: Hind limb ischemia was generated by femoral artery excision in streptozotocin-treated mice (model of type 1 diabetes), in Lepr(db/db) mice (model of type 2 diabetes), and in control (C57BL/6) mice.

Regenerative medicine in the treatment of peripheral arterial disease.

The last decade has witnessed a dramatic increase in the mechanistic understanding of angiogenesis and arteriogenesis, the two processes by which the body responds to obstruction of large conduit arteries. This knowledge has been translated into novel therapeutic approaches to the treatment of peripheral arterial disease, a condition characterized by progressive narrowing of lower extremity arteries and heretofore solely amenable to surgical revascularization. Clinical trials of molecular, genetic, and cell-based treatments for peripheral artery obstruction have generally provided encouraging results.

Endothelial nitric oxide synthase in human intestine resected for necrotizing enterocolitis.

Objective: To determine the expression and function of endothelial nitric oxide synthase (eNOS) in submucosal arterioles harvested from human intestine resected for necrotizing enterocolitis (NEC) or congenital bowel disease.

Study Design: eNOS expression was determined by using immunohistochemistry. The arteriolar diameter was measured in vitro at pressures of 10 to 40 mm Hg and also in response to the eNOS agonist acetylcholine (ACh), the exogenous nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine, and the smooth muscle relaxant papaverine.

IL-1beta alters hemodynamics in newborn intestine: role of endothelin.

Studies were carried out to determine the effects of IL-1beta on newborn intestinal hemodynamics. IL-1beta increased the release of ET-1 by primary endothelial cells in a dose-dependent manner; as well, it reduced expression of the endothelin (ET) type B (ET(B)) receptor on endothelial cells and increased expression of the ET type A (ET(A)) receptor on vascular smooth muscle cells. IL-1beta increased endothelial cell endothelial nitric oxide (NO) synthase (eNOS) expression but did not enhance eNOS activity as evidenced by release of NO(x) into conditioned medium in response to acetylcholine or shear stress.

Intestinal O2 consumption in necrotizing enterocolitis: role of nitric oxide.

We tested the hypothesis that inducible isoform of nitric oxide synthase (iNOS)-derived nitric oxide (NO) inhibits oxygen consumption (VO2) in human intestine resected for necrotizing enterocolitis (NEC). Each NEC resection specimen was divided into two sections based on histologic appearance: healthy or diseased. Intestine removed from infants for reasons other than NEC was used as control.

The effect of phenotype on mechanical stretch-induced vascular smooth muscle cell apoptosis.

The present study evaluated mechanical stretch-induced apoptosis in swine vascular smooth muscle cells (VSMC) of different phenotypes. We demonstrated that differentiated VSMC express a greater level of Bcl-2-associated death factor (BAD) and have a significant cell loss when exposed to mechanical stretch (10% elongation, 1 Hz) for 24 h. We further demonstrated that apoptosis was significantly increased only in differentiated VSMC exposed to mechanical stretch.

Ischemia and necrotizing enterocolitis: where, when, and how.

While it is accepted that ischemia contributes to the pathogenesis of necrotizing enterocolitis (NEC), three important questions regarding this role subsist. First, where within the intestinal circulation does the vascular pathophysiology occur? It is most likely that this event begins within the intramural microcirculation, particularly the small arteries that pierce the gut wall and the submucosal arteriolar plexus insofar as these represent the principal sites of resistance regulation in the gut. Mucosal damage might also disrupt the integrity or function of downstream villous arterioles leading to damage thereto; thereafter, noxious stimuli might ascend into the submucosal vessels via downstream venules and lymphatics.

Endothelin-1 in human intestine resected for necrotizing enterocolitis.

Objectives: We asked if the tissue concentration of the potent vasoconstrictor endothelin-1 (ET-1) is greater in areas of human preterm intestine that demonstrate histologic evidence of necrotizing enterocolitis (NEC) when compared with relatively healthy areas within the same resection specimen. We then evaluated if ET-1 participates in hemodynamic regulation within intestinal subserosal arterioles harvested from portions of human preterm intestine that demonstrate NEC.

Study Design: Human preterm intestine resected for NEC was divided into three zones based on proximity to the perforation (zone 1 most proximal, zone 3 most distal).

Development of the myogenic response in postnatal intestine: role of PKC.

Previous attempts to determine developmental changes in the vascular myogenic response have been confounded by the presence of competing vasoactive stimuli or the use of isolated vessels with markedly different baseline diameters. To circumvent these issues, small mesenteric arteries (diameter approximately 150 microm) from 1- and 10-day-old piglets were studied in vitro under no-flow conditions. In situ studies demonstrated that the intravascular pressure and diameter of these vessels were similar in both age groups, allowing an effective comparison of the myogenic response not obscured by differences in basal diameter.

Presentation of nitric oxide regulates monocyte survival through effects on caspase-9 and caspase-3 activation.

In the absence of survival factors, blood monocytes undergo spontaneous apoptosis, which involves the activation of caspase-3. Although nitric oxide can block caspase-3 activation and promote cell survival, it can also induce apoptosis. We hypothesized that nitrosothiols that promote protein S-nitrosylation would reduce caspase-3 activation and cell survival, whereas nitric oxide donors (such as 1-propamine 3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA) NONOate and diethylamine (DEA) NONOate) that do not target thiol residues would not.

Developmental expression of eNOS in postnatal swine mesenteric artery.

Developmental changes in the expression of endothelial nitric oxide synthase (eNOS) within the mesenteric artery of swine were studied in fetal (110 days postconception/117 days total gestation) and on postnatal days 1, 3, 10, and 30. Subjects in the 1-day-old group were subdivided into fed and nonfed. Transcription of eNOS was determined by real-time PCR, protein expression was evaluated by Western blotting, and hemodynamic and oxygenation parameters were measured within in situ gut loops before and after the administration of N(G)-monomethyl-L-arginine (L-NMMA).

Newborn intestinal circulation. Physiology and pathophysiology.

The physiologic characteristics of the newborn intestinal circulation are unique when compared with the adult condition. Most important, intestinal vascular resistance across newborn intestine is exceptionally low and this transient reduction is mediated by an increased constitutive and stimulated production of NO. The low vascular resistance characteristic of newborn intestine alters the capacity of this vasculature to respond to systemic circulatory perturbations, such as hypotension and arterial hypoxemia.