eNOS-KO Mice Research Articles

High-salt intake enhances superoxide activity in eNOS knockout mice leading to the development of salt sensitivity

A deficiency in nitric oxide (NO) generation leads to salt-sensitive hypertension, but the role of increased superoxide (O(2)(-)) in such salt sensitivity has not been delineated. We examined the hypothesis that an enhancement in O(2)(-) activity induced by high-salt (HS) intake under deficient NO production contributes to the development of salt-sensitive hypertension. Endothelial NO synthase knockout (eNOS KO; total n = 64) and wild-type (WT; total n = 58) mice were given diets containing either normal (NS; 0.4%) or high-salt (HS; 4%) for 2 wk. During this period, mice were chronically treated with a O(2)(-) scavenger, tempol (400 mg/l), or an inhibitor of NADPH oxidase, apocynin (1 g/l), in drinking water or left untreated (n = 6-8 per group). Blood pressure was measured by radiotelemetry and 24-h urine samples were collected in metabolic cages. Basal mean arterial pressure (MAP) in eNOS KO was higher (125 +/- 4 vs. 106 +/- 3 mmHg) compared with WT. Feeding HS diet did not alter MAP in WT but increased it in eNOS KO to 166 +/- 9 mmHg. Both tempol and apocynin treatment significantly attenuated the MAP response to HS in eNOS KO (134 +/- 3 and 139 +/- 4 mmHg, respectively). Basal urinary 8-isoprostane excretion rates (U(Iso)V), a marker for endogenous O(2)(-) activity, were similar (2.8 +/- 0.2 and 2.4 +/- 0.3 ng/day) in both eNOS KO and WT mice. However, HS increased U(Iso)V more in eNOS KO than in WT (4.6 +/- 0.3 vs. 3.8 +/- 0.2 ng/day); these were significantly attenuated by both tempol and apocynin treatment. These data indicate that an enhancement in O(2)(-) activity contributes substantially to the development of salt-sensitive hypertension under NO-deficient conditions.

Endothelial von Willebrand Factor Release Due to eNOS Deficiency Predisposes to Thrombotic Microangiopathy in Mouse Aging Kidney

Endothelial dysfunction is critical in the decline of renal function with. By using endothelial nitric oxide synthase knockout (eNOSKO) mice, we tested the hypothesis that a lack of endothelial nitric oxide synthase accelerates renal injury in the aging kidney. In contrast to control mice and young eNOSKO mice, aging eNOSKO mice showed greater renal injury and in particular developed a thrombotic microangiopathy, with mesangiolysis, endothelial swelling, endothelial cell loss, double-contour appearance of glomerular basement membrane (GBM), and thrombus formation. Thrombi, which were composed of fibrin, platelets, and von Willebrand factor (vWF), were identified predominantly in glomerular capillaries and rarely in arterioles, but not in larger vessels. In the tubulointerstitium, tubular degeneration and macrophage infiltration were also prominent in aging eNOSKO mice. Intraluminal vWF deposition was accompanied with thrombus formation, whereas mesangial deposition of vWF was associated with mesangial matrix expansion. Furthermore, the mesangial vWF deposition was detectable in young eNOSKO mice in which severe glomerular injury had not yet developed. Finally, a higher level of serum P-selectin in eNOSKO mice was consistent with the vWF behavior and suggested exocytosis of the Weibel-Palade body by the endothelium. In conclusion, a lack of endothelial nitric oxide synthase resulted in the development of glomerular thrombotic microangiopathy. A lack of nitric oxide likely contributed to the release of vWF, leading to thrombus formation in this model.

Endothelial Nitric Oxide Synthase Deficiency Causes Collateral Vessel Rarefaction and Impairs Activation of a Cell Cycle Gene Network During Arteriogenesis

The collateral circulation is tissue- and life-saving in obstructive arterial disease. Disappointing outcomes in clinical trials aimed at augmenting collateral growth highlight the need for greater understanding of collateral biology. The role of endothelial nitric oxide synthase (eNOS) in forming native (preexisting) collaterals and remodeling in obstructive disease are unknown or controversial issues, respectively. We compared the native collateral circulation in healthy tissue and collateral remodeling after femoral artery ligation (FAL) in wild-type and eNOS-knockout (KO) mice. Perfusion after FAL fell further in adult eNOS-KOs, in association with fewer native collaterals in hindlimb (confirmed in brain). This was not attributable to impaired collateral formation in the embryo-neonate, but rather from collateral loss during growth to adulthood. Compared to wild-type, eNOS-KOs evidenced reduced collateral remodeling, angiogenesis, and flow-mediated dilation of the arterial bed supplying the collaterals, resulting in lower perfusion and greater ischemic injury at all time points over 21 days following FAL. To probe the mechanism for impaired remodeling, we performed genome-wide expression profiling of isolated, remodeling hindlimb collaterals 24 hour after FAL. Upregulation of genes encoding cytokines/chemokines, inflammatory, stress response, and cell cycle proteins was evident in wild-type mice. In contrast, expression was lower in 40 of 44 cell cycle genes in eNOS-KO mice, in association with impaired proliferation of vascular wall cells. Our findings suggest a novel role for eNOS in maintaining native collateral density during natural growth to adulthood and in collateral remodeling in obstructive disease, the latter through regulation of cell proliferation.

Neuronal Nitric Oxide Synthase in Epidermis Is Involved in Cutaneous Circulatory Response to Mechanical Stimulation

The source of nitric oxide (NO) in the cutaneous circulation remains controversial. We hypothesized that epidermis might generate NO in response to mechanical stimulation. In hairless mouse (HR-1) skin organ culture, mechanical stimulation resulted in NO release, which declined within 30 minutes after cessation. A similar NO release occurred in a reconstructed skin model containing only keratinocytes and fibroblasts and was suppressed after detachment of the epidermal layer. Moreover, the stimulation-induced NO release was significantly lower in skin organ culture from neuronal NO synthase knockout (nNOS-KO) mice, compared with wild-type (WT) mice. Mechanical stimulation of skin organ cultures from HR-1, nNOS-KO, endothelial NOS-KO (eNOS-KO), and WT mice caused an enlargement of cutaneous lymphatic vessels. The enlargement was significantly lower after detachment of the epidermal layer than in normal skin samples and was significantly lower for nNOS-KO than for WT mice. Skin blood flow in nNOS-KO mice after stimulation was significantly lower than in WT mice. eNOS-KO mice also showed lower responses than WT mice, and the difference was similar to that in the case of nNOS-KO mice. These results are consistent with the idea that NO generated by epidermal nNOS has a significant role in the cutaneous circulatory response to mechanical stimulation.

ENOS Knockout Mice with Advanced Diabetic Nephropathy Have Less Benefit from Renin-Angiotensin Blockade than from Aldosterone Receptor Antagonists

While blockade of the renin angiotensin system (RAS) is beneficial in treating many patients with diabetic nephropathy, some patients show a poor response. We hypothesized that the poor response of RAS blockade is attributed to inability to stimulate endothelial nitric oxide. Recently, we reported that diabetic eNOS knockout (KO) mice develop advanced diabetic nephropathy similar to human disease. Here, we tested the hypothesis that blockade of the RAS would be less beneficial in this model than in diabetic wild-type mice. Both enalapril and telmisartan were less effective at reducing renal injury in diabetic eNOSKO mice compared with diabetic wild-type mice. Blood pressure was only transiently reduced by these treatments in diabetic eNOSKO mice and later returned to levels similar to that of untreated diabetic eNOSKO mice. Serum aldosterone tended to be paradoxically higher with enalapril or telmisartan in diabetic eNOSKO mice, whereas these treatments tended to lower aldosterone in diabetic wild-type mice. The pathogenic role of aldosterone was demonstrated by the evidence that spironolactone significantly reduced blood pressure and prevented renal injury. In addition, a higher dose of enalapril also failed to prevent hypertension and renal injury in diabetic eNOSKO mice. In conclusion, an impaired endothelial NO response could lessen the benefit of RAS inhibition in diabetic renal disease. Aldosterone blockade may provide superior protection in this setting.

Variations of brain endothelial nitric oxide synthase concentration in rat and mouse cortex

No information exists on the differences of eNOS concentration in brain tissue, [eNOS] br, between animals during normal and hypotensive blood pressure and both between and within animals during moderate hypotension. To address these questions, we modified a commercially available enzyme-linked immunosorbent assay (ELISA) kit for determining murine [eNOS] br since no method exists to measure [eNOS] br. Optimization of the kit ELISA procedure using brain cortex homogenates from 3 normotensive rats and 1 wild-type and 1 eNOS −/− (ko) mouse included recovery evaluation for each sample and the use of an “eNOS-free” homogenate calibrator diluent obtained from a mutant eNOS-ko mouse. Initial spike-and-recovery values of 12.5–27% suggesting a substantial sample matrix effect were improved with lipid removal treatment to 37.3% and to 70% with 1:20 dilution of the sample. Calibration standards prepared using eNOS-free buffer increased recovery values to 78% in micro-punch samples. The optimized ELISA was used in micro-punch (<1 mg) brain cortex samples from 6 hypotensive rats. Whole brain [eNOS] br varied considerably from 5–11 fmol/mg wet weight and was different between normo- and hypotensive animals ( p = 0.023). The variability of [eNOS] br due to moderate hypotension in micro-punch rat brain cortex samples was composed of both between (24%) and within (76%) animal components. The differences and variability of [eNOS] br between normo- and hypotensive animals, and between and within hypotensive animals suggests the potential utility of its measurement for investigations of cerebrovascular physiology and that [eNOS] br itself could be an important factor in cerebrovascular regulation.

Role for endothelial nitric oxide synthase in nitrite-induced protection against renal ischemia–reperfusion injury in mice

Nitrite is protective against renal ischemia/reperfusion injury (IRI); an effect due to its reduction to nitric oxide (NO). In addition to other reductase pathways, endothelial NO synthase (eNOS) may also facilitate nitrite reduction in ischemic environments. We investigated the role of eNOS in sodium nitrite (60 μM, 10 ml/kg applied topically 1 min before reperfusion)-induced protection against renal IRI in C57/BL6 wild-type (WT) and eNOS knockout (eNOS KO) mice subjected to bilateral renal ischemia (30 min) and reperfusion (24 h). Markers of renal dysfunction (plasma [creatinine] and [urea]), damage (tubular histology) and inflammation (cell recruitment) were elevated following IRI in WT mice; effects significantly reduced following nitrite treatment. Chemiluminescence analysis of cortical and medullary sections of the kidney demonstrated rapid (within 1 min) distribution of nitrite following application. Whilst IRI caused a significant (albeit substantially reduced compared to WT mice) elevation of markers of renal dysfunction and damage in eNOS KO mice, the beneficial effects of nitrite were absent or reduced, respectively. Moreover, nitrite treatment enhanced renal dysfunction in the form of increased plasma [creatinine] in eNOS KO mice. Confirmation of nitrite reductase activity of eNOS was provided by demonstration of nitrite (100 μM)-derived NO production by kidney homogenates of WT mice, that was significantly reduced by l-NMMA. l-NMMA was without effect using kidney homogenates of eNOS KO mice. These results support a role for eNOS in the pathways activated during renal IRI and also identify eNOS as a nitrite reductase in ischemic conditions; activity which in part underlies the protective effects of nitrite.

Abstract 5600: GTP Cyclohydrolase/BH4 Pathway Protects EPC Function via Suppressing Oxidative Stress and Thrombospondin-1 in Salt-Sensitive Hypertension

Background: Endothelial progenitor cells (EPC) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. eNOS critically regulates EPC mobilization and function, but is uncoupled in DOCA-salt hypertension due to reduced essential cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 synthesis, protects EPC function in DOCA-salt mice. Methods and Results: EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), DOCA-salt treated Tg-GCH, and BH4 deficient hph-1 mice. Both EPC adhesion and angiogenic functions were impaired in WT DOCA (58 and 38%, respectively, n=5– 8, p&lt;0.01) and hph-1 mice (40 and 31%, respectively, n=5–8, p&lt;0.01, vs. WT), which were rescued in DOCA Tg-GCH mice (93 and 92%, respectively, n=6–7). Superoxide (O 2 − , DHE-) and NO (DAF-FM flow cytometry) levels in EPCs were elevated and reduced, respectively, in WT DOCA (220% and 53%, respectively, n=5–12, p&lt;0.01) and hph-1 mice (280% and 41%, respectively, n=6–12, p&lt;0.05, vs. WT), which were blunted in DOCA Tg-GCH mice (137% and 96%, respectively, n=6–7). Thrombospondin-1 (TSP-1), a secreted potent anti-angiogenic protein, was elevated in EPCs of WT DOCA and hph-1 mice (290 and 280%, respectively, n=8–12, p&lt;0.01 vs. WT), but not in DOCA Tg-GCH mice (160%, n=9–12, p&gt;0.05). In vitro treatment of EPCs from WT DOCA mice with BH4, PEG-SOD, or L-NNA significantly reduced TSP-1 (51, 42 and 53%, respectively, n=5–12, p&lt;0.01) and O 2 − levels (58, 67 and 69%, respectively, n=5– 6, p&lt;0.01 vs. WT-DOCA). NO level in EPCs of WT DOCA mice was rescued by BH4 and PEG-SOD (144 and 165%, respectively, n=4–7, p&lt;0.05 vs. control), but not L-NNA. Crossing eNOS KO and Tg-GCH mice resulted in preserved circulating EPC number (88%, n=4–15, p&gt;0.05 vs. WT), reduced EPC O 2 − level (64%, n=4 – 8, p&lt;0.05 vs. WT-DOCA), and improved EPC adhesion and angiogenic functions (160 and 158%, respectively, n=4 –12, p&lt;0.05 vs. WT-DOCA). Conclusion: We demonstrate for the first time that GTPCH/BH4 pathway protects EPC function via suppressing oxidative stress and TSP-1 level in salt-sensitive hypertension. This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota &amp; Wisconsin).

Hemodynamic and Histological Effects of Traumatic Brain Injury in eNOS-Deficient Mice

Microvascular dysfunction in the brain, characterized by vasoconstriction, vascular occlusion, and disruption of the blood brain barrier, may adversely affect outcome following traumatic brain injury (TBI). Because of its vasodilating and antiaggregative properties, nitric oxide (NO) produced by nitric oxide synthase in the endothelium (eNOS) is a key regulator of vascular homeostasis. The objective of the present study was to evaluate the role of eNOS in vascular disturbances and histological outcome in the brain following TBI. Cortical blood flow ([(14)C]-iodoantipyrine technique), number of perfused capillaries (FITC-dextran technique), brain water content (wet vs. dry weight), and the transfer constant (K(i)) for [(51)Cr]-EDTA, reflecting permeability, were analyzed 3 h and 24 h after a controlled cortical impact injury (CCI) in eNOS-deficient (eNOS-KO) and wild-type (WT) mice. Cortical contusion volume and cell count in the hippocampus were evaluated 3 weeks after injury. Blood flow in the injured cortex decreased in both groups following trauma. There were no significant differences between the groups at 3 h, but blood flow was lower in eNOS-KO mice than in WT mice 24 h after trauma. Brain water content was higher in the WT mice than in eNOS-KO mice at 24 h. Number of perfused capillaries, K(i), and histological outcome were similar in both groups. We conclude that eNOS is important for maintenance of cerebral blood flow after trauma and that eNOS promotes edema formation by mechanisms other than increased permeability. The vascular effects of eNOS do not, however, influence histological outcome.

Nitric oxide synthase isoforms play distinct roles during acute peritonitis

Background. Acute peritonitis is the most frequent complication of peritoneal dialysis (PD). Increased nitric oxide (NO) release by NO synthase (NOS) isoforms has been implicated in acute peritonitis, but the role played by the NOS isoforms expressed in the peritoneum is unknown.Methods. We investigated the structural and functional consequences of acute peritonitis induced by LPS in wild-type (WT) mice versus knockout mice (KO) for the endothelial NOS (eNOS), the inducible NOS (iNOS) or the neuronal NOS (nNOS).Results. The level of NO metabolites (NOx) in the dialysate was maximal 18 h after LPS injection. LPS induced a significant increase in the transport of small solutes and decreased ultrafiltration in WT mice. These changes, which occurred without vascular proliferation, were paralleled by the upregulation of nNOS and eNOS, and the induction of iNOS. The transport modifications induced by LPS were significantly reversed in eNOS KO mice, but not modified in mice lacking iNOS or nNOS. In contrast, the increase of dialysate NOx was abolished in iNOS KO mice and significantly reduced in eNOS KO mice, but left unchanged in mice lacking nNOS. Mice lacking iNOS also showed more severe inflammatory changes, and a trend towards increased mortality following LPS.Conclusion. These data demonstrate specific roles for NOS isoforms in the peritoneal membrane and suggest that selective eNOS inhibition may improve peritoneal transport during acute peritonitis.

Early phase of ischemic preconditioning in the mouse: effects of endothelial nitric oxide synthase (eNOS), ischemic duration, aging, and gender

Ischemic preconditioning (IPC) is a powerful phenomenon that provides effective cardioprotection in mammalian hearts, however, little is known in relation to prolonged ischemia (I), aging and gender. Importantly, the role of eNOS‐mediated nitric oxide (NO) pathway is still controversial. Therefore, we performed an extensive study in wild‐type (WT) and eNOS‐knockout (eNOS‐KO) mice to evaluate whether the infarct limiting effect of IPC depends on eNOS, ischemic periods, ages (12‐16 weeks and 12‐16 months), and gender. Classical IPC was induced by 3‐cycles of 5‐min regional coronary I separated by 5‐min reperfusion (R), and was followed by 30‐min or 60‐min of index I and 24‐hrs R. Control IR protocol had 30‐ or 60‐min I followed by 24‐hrs R. Protection was evaluated by measuring myocardial infarct size as a percentage of area at risk. The major findings are that regardless of age and gender, WT mice exhibited robust IPC effects with significantly smaller myocardial infarction, whereas, eNOS‐KO mice did not. In general, female WT mice had smaller infarction compared to male WT. Importantly, aging and prolonged I caused significantly larger infarction in both genders. Prolonged myocardial ischemia was fatal in eNOS‐KO mice. In conclusion, IPC protects WT mice against in vivo myocardial IR injury regardless of age, gender and/or ischemic duration, but, deletion of eNOS abolishes the cardioprotective effect of early IPC.

ENOS signaling is essential in GLP‐2‐mediated stimulation of blood flow, but not cell proliferation in the mouse gut

Through a G protein‐coupled receptor, glucagon‐like peptide‐2 (GLP‐2) stimulates intestinal crypt cell proliferation and mucosal blood flow. GLP‐2 receptor is localized to enteric neurons, endocrine cells, and myofibroblasts, but not enterocytes. However, it is largely unknown how GLP‐2 receptor‐activated mitogenic, cytoprotective, and vasoactive signals are conveyed to the intestinal epithelium. Nitric oxide can function as a cellular signal to regulate cell growth and blood flow. Thus, our objective was to determine whether nitric oxide is a key mediator in the GLP‐2 receptor‐activated enteric signaling network. Endothelial nitric oxide synthase knockout (eNOS KO) and wild‐type (eNOS WT) mice (n=40) were subcutaneously injected with GLP‐2 or saline for 14 d, and with BrdU 2 h prior to euthanization. Mucosal cell proliferation, apoptosis, and blood flow were determined by BrdU‐positive cells, active caspase‐3 abundance, and hemoglobin (Hb) content, respectively. There was no difference between eNOS KO and WT mice in gut growth (indicated by mucosal weight, protein mass, villous height and crypt depth), cell proliferation, apoptosis, or blood flow. GLP‐2 increased gut growth by stimulating crypt cell proliferation in both KO and WT mice. GLP‐2 appeared to decrease apoptosis in the WT mice, but increase it in the KO mice. Importantly, GLP‐2 increased gut blood flow in the WT mice, but not in the KO mice. We conclude that GLP‐2‐induced vasoactive action is mediated by the eNOS signaling, but GLP‐2‐stimulated tropic action is independent of it. (Supported by USDA CRIS 6250‐51000‐043 and NIDDK K01DK75489)

Lowering Blood Pressure Blocks Mesangiolysis and Mesangial Nodules, but Not Tubulointerstitial Injury, in Diabetic eNOS Knockout Mice

Recently, we and others reported that diabetic endothelial nitric oxide synthase knockout (eNOSKO) mice develop advanced glomerular lesions that include mesangiolysis and nodular lesions. Interestingly, insulin treatment lowered blood pressure and prevented renal lesions, raising the question as to whether these beneficial effects of insulin were due to its ability to lower either high glucose levels or high blood pressure. We, therefore, examined the effect of lowering blood pressure using hydralazine in this diabetic eNOSKO mouse model. Hydralazine treatment significantly blocked the development of mesangiolysis and microaneurysms, whereas tubulointerstitial injury was not prevented in these mice. Additionally, hydralazine did not reduce expression levels of either tubulointerstitial thrombospondin-1 or transforming growth factor-beta despite controlling blood pressure. On the other hand, the critical role of high glucose levels on the development of tubulointerstitial injury was suggested by the observation that serum glucose levels were correlated with tubulointerstitial injury, as well as with the expression levels of both transforming growth factor-beta and thrombospondin-1. Importantly, controlling blood glucose with insulin completely blocked tubulointerstitial injury in diabetic eNOSKO mice. These data suggest that glomerular injury is dependent on systemic blood pressure, whereas hyperglycemia may have a more important role in tubulointerstitial injury, possibly due to the stimulation of the thrombospondin-1-transforming growth factor-beta pathway in diabetic eNOSKO mice. This study could provide insights into the pathogenesis of advanced diabetic nephropathy in the presence of endothelial dysfunction.

I022 Role of heme oxygenase-1 in high blood flow (shear stress)-induced remodeling of rat resistance arteries

We have previously shown that high blood flow (HF)-induced remodeling in mesenteric resistance arteries (MRA) failed to occur in aging. Heme oxygenase (HO)-1 is known to be induced by hemodynamic forces in VSM and endothelial cells. The aim of this work was to evaluate: i) the physiological role of HO activity in HF remodeling in MRA, and ii) the effect of systemic HO-1 induction on altered HF remodeling in MRA of aged rats. First-order MRA from 3- and 18-months-old rats were exposed to HF for 2 (young) or 14 days (young and old) by alternate ligation of 2nd-order arteries. I. Outward hypertrophic remodeling in HF arteries of young control rats (increased diameter and medial cross-sectional area) was associated with a profound increase in the expression of HO-1 mRNA two days after the ligation. II. HO inhibition with Sn-protoporphyrin (50 μmol/kg/day, i.p.) significantly inhibited diameter enlargement in HF arteries. III. HO-1 protein was barely detected in MRA from both young and old rats but was markedly increased after treatment with the HO-1 inducer Co-protoporphyrin (CoPP, 5 mg/kg, twice a week, s.c.). In old rats, CoPP decreased mean arterial blood pressure to a level similar to that in young animals (80±2.5 vs 101.5±2 mmHg, compared with untreated old rats) suggesting that CoPP may act as vasodilator. Moreover, HO-1 induction restored HF remodeling in old animals (17±2 vs 3±3 % increase in diameter, and 18440±2192 vs 13805±323μm2 for medial cross-sectional area when compared to HF in control old rats ; pressure=75mmHg). IV. This latter data was supported by the ability of CoPP to restore HF remodeling in eNOS-KO mice. These novel data show the pivotal role of HO activity in HF-induced remodeling in MRA. This likely occurs via its action on diameter enlargement in parallel with MMPs-dependent matrix degradation, downstream eNOS. Thus, selective HO-1 induction can be used as a new therapeutic approach to ameliorate the prognosis of cardiovascular injury associated with altered adaptation capacity of small arteries to chronic changes in local blood flow such as in ischemic and metabolic diseases or in hypertension, more frequent in the elderly.

Endothelial-Specific Expression of Mitochondrial Thioredoxin Promotes Ischemia-Mediated Arteriogenesis and Angiogenesis

Thioredoxin-2 (Trx2), a major antioxidant protein in mitochondria, enhances nitric oxide bioavailability and inhibits ASK1-dependent apoptosis in endothelial cells (ECs). However, the in vivo role of Trx2 in angiogenesis has not been defined. Here we used EC-specific transgenesis of Trx2 (Trx2-TG) in mice to determine the in vivo function of Trx2 in arteriogenesis and angiogenesis. In a femoral artery ligation model, Trx2-TG mice had enhanced capacity in limb perfusion recovery and ischemic reserve capacity compared to the nontransgenic littermates. Ischemia-initiated arteriogenesis in the upper limb was augmented in Trx2-TG mice. Trx2-TG mice also showed significantly enhanced capillary formation and maturation in the lower limb. In nontransgenic limb, ischemia specifically induced a downregulation of Trx2 protein, leading to increased oxidative stress, ASK1 activation, and EC apoptosis. In contrast, Trx2-TG maintained a constitutive level of Trx2, reducing the ischemia-induced deleterious responses. We then defined the mechanism by which Trx2 increases angiogenesis using ECs isolated from Trx2-TG mice. Trx2-TG ECs showed increased NO and NO-dependent migration. In addition, these cells were more resistant to oxidative stress-induced activation of ASK1 signaling and apoptosis. Moreover, Trx2-augmented EC survival is NO-independent. To define the relative contributions of Trx2-increased NO and Trx2-reduced ASK1 apoptotic activity to angiogenesis in vivo, we examined Trx2 effects on ischemia-induced angiogenesis in eNOS-deficient mice. The eNOS deletion caused severe impairment in the functional flow recovery in response to ischemia. Trx2 expression in eNOS-KO mice still dramatically inhibited ischemia-induced ASK1 and EC apoptosis, leading to an enhanced functional flow recovery. These in vivo and in vitro data support that Trx2 maintains EC function by two parallel pathways-scavenging ROS to increase NO bioavailability and inhibiting ASK1 activity to enhance EC survival, facilitating ischemia-mediated arteriogenesis and angiogenesis.

Caloric Restriction Stimulates Revascularization in Response to Ischemia via Adiponectin-mediated Activation of Endothelial Nitric-oxide Synthase

Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, the functional roles of CR in regulation of revascularization in response to ischemia have not been examined. Here we investigated whether CR modulates vascular response by employing a murine hindlimb ischemia model. Wild-type (WT) mice were randomly divided into two groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb ischemic surgery. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared with WT mice fed AL (AL-WT). Revascularization of ischemic hindlimb relative to the contralateral limb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow and capillary density analyses. CR-WT mice had significantly higher plasma levels of the fat-derived hormone adiponectin compared with AL-WT mice. In contrast to WT mice, CR did not affect the revascularization of ischemic limbs of adiponectin-deficient (APN-KO) mice. CR stimulated the phosphorylation of endothelial nitric-oxide synthase (eNOS) in the ischemic limbs of WT mice. CR increased plasma adiponectin levels in eNOS-KO mice but did not stimulate limb perfusion in this strain. CR-WT mice showed enhanced phosphorylation of AMP-activated protein kinase (AMPK) in ischemic muscle, and administration of AMPK inhibitor compound C abolished CR-induced increase in limb perfusion and eNOS phosphorylation in WT mice. Our observations indicate that CR can promote revascularization in response to tissue ischemia via an AMPK-eNOS-dependent mechanism that is mediated by adiponectin.

Abstract 3651: Caloric Restriction Stimulates Ischemia-induced Angiogenesis through an eNOS-dependent Pathway

Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, little is known about the role of CR in the process of ischemia-induced vascular remodeling. Here, we investigated whether CR regulates angiogenic responses in vivo employing the hindlimb model of ischemia-induced angiogenesis. Wild-type (WT), endothelial nitric oxide synthase deficient (eNOS-KO) or adiponectin deficient (APN-KO) mice were randomly divided into 2 groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb surgery. Serum adiponectin levels were measured by ELISA. The expression and activation of eNOS were assessed by Western blot analysis. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared to WT mice fed AL (AL-WT). Angiogenic repair of ischemic hindlimb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow (p&lt;0.05) and capillary density analyses (p&lt;0.05). In WT mice, CR significantly enhanced ischemia-induced increase in eNOS activation of muscle tissues and increased serum adiponectin levels. In eNOS-KO mice, CR significantly increased serum adiponectin levels but did not enhance limb perfusion after surgery. In APN-KO mice, ischemia induction did not increase eNOS activation in muscle tissues, and CR had no effects on blood flow recovery and eNOS activation in ischemic limb. Our observation indicates that CR can promote angiogenesis in response to tissue ischemia through its ability to increase eNOS activation that is possibly mediated by adiponectin.

The pivotal role of VEGF on glomerular macrophage infiltration in advanced diabetic nephropathy

A growing body of evidence implicates inflammation in the development of diabetic nephropathy. We recently reported that diabetic endothelial nitric oxide synthase knockout (eNOS KO) mice develop advanced glomerular lesions resembling human diabetic nephropathy. Vascular endothelial growth factor (VEGF) is a major factor in diabetic nephropathy, and is known to be chemotactic for macrophages. Herein, we examined the association of VEGF with macrophage infiltration in experimental diabetic nephropathy. Glomerular macrophage infiltration was markedly increased in diabetic eNOS KO mice compared to diabetic C57BL/6 mice, and correlated with glomerular injury, such as mesangiolysis, glomerular microaneurysm and nodular lesions of glomerular sclerosis. An elevation of podocyte VEGF expression correlated with infiltration of Flt-1-positive macrophage in injured glomeruli in diabetic eNOS KO mice, suggesting that VEGF could contribute to macrophage migration. Neither renal nNOS nor iNOS expression was altered in both C57BL/6 and eNOS KO mice. To determine if lack of NO could affect VEGF activation of macrophages, we examined if exogenous NO can block macrophage migration induced by VEGF in in vitro studies. Exogenous NO blocked macrophage migration and hypertrophy in response to VEGF. NO mediated these effects in part by downregulating Flt-1 expression on the macrophage. In summary, NO negatively regulates VEGF-induced macrophage migration by inhibiting Flt-1 expression. The VEGF–endothelial NO uncoupling pathway might partially explain how VEGF causes glomerular disease in diabetes.

Role of transcription factors in estrogen‐dependent upregulation of CYP 2C family in arteries of eNOS deficient mice

Upregulation of cytochrome P450 (CYP) 2C29/38/40 is responsible for the EET‐mediated flow‐dependent dilation in microvessels of female mice, as a function of NO deficiency. In the present studies, we provide evidence of the correlation between transcription factors (PPAR‐γ, PPAR‐δ and RXR‐γ) and CYP 2C29/38/40 in isolated arterioles of female eNOS‐KO mice by microarray (SuperArray, OMM‐401). Five single arterioles isolated from mesentery of both genders of eNOS‐KO and WT mice were pooled and snap frozen in liquid nitrogen; ∼10 μg of total RNA were isolated, and a total of 10 arrays from 2 WT and 3 eNOS‐KO mice of each gender were performed. Microarray results indicate that associated with a specific upregulation of CYP 2C29/38/40, expression of genes for PPAR‐γ, PPAR‐δ and RXR‐γ, the nuclear receptors that function as gene regulators via specific DNA binding, was also increased (P&lt;0.01, 0.05 and 0.03, respectively) in female eNOS‐KO compared to male eNOS‐KO vessels. These significant differences were validated by quantitative real‐time PCR and Western blot analysis. The increase in gene expression for the three transcription factors was only observed in vessels of female, but not in those of male eNOS‐KO mice, and without significant differences between vessels of WT male and WT female mice, a pattern that is similar to the upregulation of CYP 2C/29/38/40. Thus, the microarray analysis screened the global expression of genes for the transcription factors and revealed the specificity of estrogen and NO deficiency in the control of the transcription factors which may play a role in the regulation of CYP 2C expression (Supported by NIH HL‐43023, HL‐68813 and HL‐070653).

Impairment of endothelial nitric oxide synthase causes abnormal fat and glycogen deposition in liver

Nitric oxide (NO) affects fatty acid synthesis and biogenesis of fatty acid consuming mitochondria. However, whether NO generated by the endothelial NO synthase isoform (eNOS) has significant impact on the synthesis and deposition of fat in liver remained unclear. We analyzed the quantity and distribution of mitochondria and fat in liver of wild-type (WT) mice and mice lacking eNOS (eNOS-KO). The livers of eNOS-KO mice contained tenfold more fat close (zone 1) and twenty fold more distal (zone 3) to the artery. The fat was deposited as droplets co-localized with mitochondria. Additionally, the livers of eNOS-KO mice contained 1.5-fold more homogenously distributed glycogen. No difference in the quantity of mitochondria was found between liver homogenates of eNOS-KO mice and WT animals. Mitochondria from liver homogenates of eNOS-KO mice exhibited a higher ratio of citrate synthase (CS) and NADH–cytochrome c oxidoreductase (KI+III) activity. We conclude that lack of eNOS-derived NO stimulates citrate- and lipid synthesis in liver thus contributing to the development of overweight. In support of this view, more visceral fat and 70% higher body weight was determined in one year old eNOS-KO mice in comparison to WT animals.