Loss Of Endothelial Nitric Oxide Research Articles

Abstract P128: Endothelial Nitric Oxide Determines Hematopoietic Cell Fate and Osteoclastogenesis By Modulating CSF1 During Hypertension

Introduction: Loss of endothelial nitric oxide (NO) is common in hypertension (HTN). The bone marrow niche contains endothelial cells; however, how endothelial NO affects hematopoiesis and bone in HTN remains undefined. Hypothesis: We hypothesized that NO loss in HTN drives hematopoiesis in the bone marrow, later favoring osteoclastogenesis and bone resorption. Methods and Results: By microcomputed tomography, eNOS deficient mice (eNOS -/- ) had reduced femoral cortical area (Figure 1A) and trabecular bone volume fraction (Figure 1B) compared to wildtype mice (WT). Flow cytometry showed that granulocyte-monocyte (Figure 1C) and common lymphoid progenitors (Figure 1D) were markedly higher in eNOS -/- than in WT. mRNA analysis revealed loss of endothelial NO increased expression of cathepsin K (Ctsk), colony stimulating factor-1 receptor (CSF1R), and tartrate resistant acid phosphatase (TRAP) in the bone marrow. We exposed murine bone-marrow endothelial cells (BMECs) to 5% (normotension), 5% with L-NAME, or 10% (HTN) cyclic strain. Compared to 5% strain, BMECs exposed to L-NAME or 10% strain exhibited markedly increased CSF1 production, an essential cytokine for myelopoiesis and osteoclastogenesis (Figure 1E and F). In coculture experiments, we also found that macrophages cultured with BMECs undergoing 5% strain with L-NAME or 10% strain exhibited higher expression of Ctsk, CSF1R, and TRAP, indicating osteoclast formation. Conclusion: Endothelial NO likely inhibits osteoclastogenesis and bone resorption. The loss of NO through mechanical stretch or inhibiting eNOS increases CSF1 production and hematopoiesis. This shows a potential mechanism by which NO loss leads to pathological hematopoiesis and bone resorption during HTN.

Partial loss of endothelial nitric oxide leads to increased cerebrovascular beta amyloid

Cerebral amyloid angiopathy (CAA) is present in over half of the elderly population and in 80–90% of Alzheimer’s disease (AD) patients. CAA is defined by the deposition of beta amyloid (Aβ) in small cerebral arteries and capillaries. Cardiovascular risk factors are associated with an increased incidence of CAA. We utilized 18-month-old endothelial nitric oxide synthase (eNOS) heterozygous knockout (+/−) mice, a clinically relevant model of endothelial dysfunction, to examine the role of endothelial nitric oxide (NO) in vascular Aβ accumulation. eNOS+/− mice had significantly higher vascular levels of Aβ40 (P < 0.05). Aβ42 was not detected. There was no difference in Aβ in brain tissue. Amyloid precursor protein and β-site APP cleavage enzyme 1 protein levels were unaltered, while levels of the α-secretase enzyme, a disintegrin and metalloproteinase 10, were significantly lower in eNOS + /− microvascular tissue (P < 0.05). Insulin degrading enzyme and low-density lipoprotein receptor-related protein 1 were significantly increased in eNOS+/− microvascular tissue, most likely an adaptive response to locally higher Aβ concentrations. Lastly, catalase and CuZn superoxide dismutase were significantly elevated in eNOS+/− microvascular tissue (P < 0.05). These data demonstrate decreased availability of endothelial NO leads to increased cerebrovascular concentration of Aβ along with compensatory mechanisms to protect the vasculature.

Targeting Mitochondrial Deacetylase Sirt3 in Endothelial Dysfunction and Hypertension

Hypertension is a multifactorial disorder involving perturbations of the vasculature, the kidney and the central nervous system. It represents a major risk factor for stroke, myocardial infarction, and heart failure; however, despite treatment with multiple drugs, one third of hypertensive patients remain hypertensive. We have recently shown that hypertension is associated with mitochondrial hyperacetylation due to reduced activity of the key mitochondrial deacetylase Sirt3 (Dikalova et al. Circ Res. 2017;121(5):564–574.). We hypothesized that targeting Sirt3 has therapeutic potential in endothelial dysfunction and hypertension. To test this hypothesis we have developed novel tamoxifen-inducible endothelium specific Sirt3 knockout (EcSirt3KO), inducible endothelial specific Sirt3 overexpressing (EcSirt3OX) mice and global Sirt3 overexpressing (Sirt3OX) on C57Bl/6J background, and examined the effect of Sirt3 expression on vascular dysfunction and hypertension. Blood pressure was measured by telemetry. Vascular superoxide was measured by HPLC analysis of O2• specific products of DHE and mitoSOX. Vascular NO was measured by EPR and NO spin trap Fe(DETC)2. Vascular reactivity was studied using isometric tension and vascular permeability was tested by Evans Blue assay. Angiotensin II-induced hypertension was markedly increased in EcSirt3KO compared with wild-type C57Bl/6J mice. Decrease of NO is a hallmark of endothelial dysfunction in hypertension due to vascular oxidative stress. Indeed, angiotensin II infusion in EcSirt3KO mice causes severe loss of endothelial nitric oxide (3-fold vs 2-fold in wild-type), overproduction of vascular superoxide, exacerbated endothelial dysfunction and increased vascular permeability. In contrast to EcSirt3KO, Sirt3 overexpression in Sirt3OX mice significantly attenuates hypertension by 25 mm Hg, prevents overproduction of vascular superoxide, protects endothelial dependent relaxation and vascular permeability. Interestingly, endothelial specific Sirt3 overexpression in EcSirt3OX mice mimics the protective effect of global Sirt3 overexpression by blocking vascular oxidative stress, protecting vascular relaxation and attenuating angiotensin II-induced hypertension. Our studies demonstrate that endothelial Sirt3 protects vascular functions and attenuates hypertension. These data support a therapeutic potential of targeting Sirt3 in vascular dysfunction and hypertension. Support or Funding Information This work was supported by funding from National Institute of Health (R01HL124116 and PO1HL129941), Vanderbilt University (VR7040 and UL1 RR024975). Dr. Dikalova was supported by American Heart Association (16GRNT31230017) and Dr. Itani was funded by the National Institute of Health (1F32HL124972). Vascular relaxation in wild-type and Sirt3OX mice before and after angiotensin II-infusion (0.7 mg/kg/day). Vascular reactivity was studied using isometric tension (B). Blood pressure in wild-type and Sirt3OX mice. (A) Systolic blood pressure was measured by telemetry before and after angiotensin II-infusion (0.7 mg/kg/day). Insert shows Western blot of Sirt3 and SOD2 in aorta mitochondria isolated from WT and Sirt3OX mice. Aaortic superoxide in wild-type and Sirt3OX mice before and after angiotensin II-infusion (0.7 mg/kg/day) was measured by DHE/HPLC assay (C). Results are mean ± SEM (n=5). *P<0.01 vs WT. **P<0.05 vs WT+AngII. Aortic endothelial nitric oxide in wild-type and Sirt3OX mice before and after angiotensin II-infusion (0.7 mg/kg/day) was measured by Electron Spin Resonance (D). Results are mean ± SEM (n=5). *P<0.01 vs WT. **P<0.05 vs WT+AngII. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of endothelial nitric oxide synthase exacerbates intestinal and lung injury in experimental necrotizing enterocolitis

BackgroundNecrotizing enterocolitis (NEC) continues to be a devastating condition among preterm infants. Nitric oxide, which is synthesized in the intestine by endothelial nitric oxide synthase (eNOS), acts as a potent vasodilator and antioxidant within the mesentery and may play a role in prevention of NEC. We hypothesized that loss of endothelial nitric oxide would worsen both intestinal and associated lung injury and increase local and systemic inflammation during experimental NEC. MethodsNEC was induced in five-day-old wild type (WT) and eNOS-knockout (eNOSKO) mouse pups. Experimental groups (n=10) were formula fed and subjected to intermittent hypoxic and hypothermic stress, while control groups (n=10) remained with their mother to breastfeed. Pups were monitored by daily clinical assessment. After sacrifice on day nine, intestine and lung were assessed for injury, and cytokines were measured in tissue homogenates by ELISA. Data were compared with Mann–Whitney, and p<0.05 was significant. ResultsEach NEC group was compared to its respective strain’s breastfed control to facilitate comparisons between the groups. Both NEC groups were significantly sicker than their breastfed controls. eNOSKO NEC animals had a median clinical assessment score of 3 (IQR=1–5), and the WT NEC animal’s median score was 3 (IQR=2–5). Despite similar clinical scores, intestinal injury was significantly worse in the eNOSKO NEC groups compared to WT NEC groups (median injury scores of 3.25 (IQR=2.25–3.625) and 2 (IQR=1–3), respectively (p=0.0474). Associated lung injury was significantly worse in the eNOSKO NEC group as compared to the WT NEC group (median scores of 8.5 (IQR=6.75–11.25) and 6.5 (IQR=5–7.5), respectively, p=0.0391). Interestingly, cytokines in both tissues were very different between the two groups, with varying effects noted for each cytokine (IL-6, IL-1β, VEGF, and IL-12) in both tissues. ConclusionNitric oxide from eNOS plays a key role in preventing the development of NEC. Without eNOS function, both intestinal and lung injuries are more severe, and the inflammatory cascade is significantly altered. Further studies are needed to determine how eNOS-derived nitric oxide facilitates these beneficial effects.

Loss of Endothelial Nitric Oxide Synthase Promotes p25 Generation and Tau Phosphorylation in a Murine Model of Alzheimer's Disease.

Alzheimer's disease has an unknown pathogenesis; however, cardiovascular risk factors are associated with a higher incidence of Alzheimer's disease. A defining feature of endothelial dysfunction induced by cardiovascular risk factors is reduced bioavailable endothelial nitric oxide (NO). We previously demonstrated that endothelial NO acts as an important signaling molecule in neuronal tissue. We sought to determine the relationship between the loss of endothelial NO synthase (eNOS) and tau phosphorylation in neuronal tissue. We used eNOS knockout (-/-) mice as well as an Alzheimer's disease mouse model, amyloid precursor protein (APP)/PSEN1dE9+/- (PS1) that lacked eNOS (APP/PS1/eNOS-/-) to examine expression of tau kinases and tau phosphorylation. Brain tissue from eNOS-/- mice had statistically higher ratios of p25/p35, indicative of increased cyclin-dependent kinase 5 activity as compared with wild-type (n=8, P<0.05). However, tau phosphorylation was unchanged in eNOS-/- mice (P>0.05). Next, we determined the role of NO in tau pathology in APP/PS1/eNOS-/-. These mice had significantly higher levels of p25, a higher p25/p35 ratio (n=12-14; P<0.05), and significantly higher cyclin-dependent kinase 5 activity (n=4; P<0.001). Importantly, APP/PS1/eNOS-/- mice also had significantly increased tau phosphorylation (n=4-6; P<0.05). No other changes in amyloid pathology, antioxidant pathways, or neuroinflammation were observed in APP/PS1/eNOS-/- mice as compared with APP/PS1 mice. Our data suggests that loss of endothelial NO plays an important role in the generation of p25 and resulting tau phosphorylation in neuronal tissue. These findings provide important new insights into the molecular mechanisms linking endothelial dysfunction with the pathogenesis of Alzheimer's disease.

Increased pulmonary arteriolar tone associated with lung oxidative stress and nitric oxide in a mouse model of Alzheimer's disease.

Vascular dysfunction and decreased cerebral blood flow are linked to Alzheimer's disease (AD). Loss of endothelial nitric oxide (NO) and oxidative stress in human cerebrovascular endothelium increase expression of amyloid precursor protein (APP) and enhance production of the Aβ peptide, suggesting that loss of endothelial NO contributes to AD pathology. We hypothesize that decreased systemic NO bioavailability in AD may also impact lung microcirculation and induce pulmonary endothelial dysfunction. The acute effect of NO synthase (NOS) inhibition on pulmonary arteriolar tone was assessed in a transgenic mouse model (TgAD) of AD (C57BL/6‐Tg(Thy1‐APPSwDutIowa)BWevn/Mmjax) and age‐matched wild‐type controls (C57BL/6J). Arteriolar diameters were measured before and after the administration of the NOS inhibitor, L‐NAME. Lung superoxide formation (DHE) and formation of nitrotyrosine (3‐NT) were assessed as indicators of oxidative stress, inducible NOS (iNOS) and tumor necrosis factor alpha (TNF‐α) expression as indicators of inflammation. Administration of L‐NAME caused either significant pulmonary arteriolar constriction or no change from baseline tone in wild‐type (WT) mice, and significant arteriolar dilation in TgAD mice. DHE, 3‐NT, TNF‐α, and iNOS expression were higher in TgAD lung tissue, compared to WT mice. These data suggest L‐NAME could induce increased pulmonary arteriolar tone in WT mice from loss of bioavailable NO. In contrast, NOS inhibition with L‐NAME had a vasodilator effect in TgAD mice, potentially caused by decreased reactive nitrogen species formation, while significant oxidative stress and inflammation were present. We conclude that AD may increase pulmonary microvascular tone as a result of loss of bioavailable NO and increased oxidative stress. Our findings suggest that AD may have systemic microvascular implications beyond central neural control mechanisms.

Neurovascular Protective Function of Endothelial Nitric Oxide - Recent Advances.

In the central nervous system endothelial nitric oxide (NO) is an essential molecule responsible for the preservation of the functional integrity of the neurovascular unit. NO causes vasodilatation and is an important inhibitor of platelet aggregation, smooth muscle cell proliferation, and white blood cell adhesion. In addition, endothelium-derived NO exerts anti-inflammatory and pro-angiogenic effects. More recently, it has been recognized that endothelial NO modulates the expression and processing of amyloid precursor protein in cerebrovascular endothelium and neuronal tissue. Studies in endothelial NO synthase (eNOS) knockout mice indicate that endothelial NO functions as a neurovascular protective molecule during aging. Indeed, genetic inactivation of eNOS exacerbates the detrimental effects of aging on cerebrovascular, microglial, and neuronal functions as well as on cognition. These findings suggest that the preservation of healthy endothelium and normal function of eNOS might be important therapeutic targets. Because the beneficial effects of NO are mostly mediated by the activation of guanylate cyclase/cyclic GMP signaling, inhibitors of phosphodiesterase isoforms, or activation of this signaling with exercise, may offer therapeutic opportunities in the prevention and treatment of aging-induced cognitive decline and Alzheimer's disease. Most recent advances in understanding the molecular mechanisms linking loss of endothelial NO with cognitive decline will be discussed in this review. (Circ J 2016; 80: 1499-1503).

Endothelial nitric oxide: protector of a healthy mind

Endothelial nitric oxide (NO) is generated by constitutively active endothelial nitric oxide synthase (eNOS), an essential enzyme responsible for cardiovascular homeostasis. Historically, endothelial NO was first recognized as a major vasodilator involved in control of vasomotor function and local blood flow. In this review, our attention is focused on the emerging role of endothelial NO in linking cerebrovascular function with cognition. We will discuss the recognized ability of endothelial NO to modulate processing of amyloid precursor protein (APP), influence functional status of microglia, and affect cognitive function. Existing evidence suggests that the loss of NO in cultured human cerebrovascular endothelium causes increased expression of APP and β-site APP-cleaving enzyme 1 (BACE1) thereby resulting in increased secretion of amyloid β peptides (Aβ1-40 and Aβ1-42). Furthermore, increased expression of APP and BACE1 as well as increased production of Aβ peptides was detected in the cerebral microvasculature and brain tissue of eNOS-deficient mice. Since Aβ peptides are considered major cytotoxic molecules responsible for the pathogenesis of Alzheimer's disease, these observations support the concept that a loss of endothelial NO might significantly contribute to the initiation and progression of cognitive decline. In addition, genetic inactivation of eNOS causes activation of microglia and promotes a pro-inflammatory phenotype in the brain. Behavioural analysis revealed that eNOS-deficient mice exhibit impaired cognitive performance thereby indicating that selective loss of endothelial NO has a detrimental effect on the function of neuronal cells. Together with findings from prior studies demonstrating the ability of endothelial NO to affect synaptic plasticity, mitochondrial biogenesis, and function of neuronal progenitor cells, it is becoming apparent that the role of endothelial NO in the control of central nervous system function is very complex. We propose that endothelial NO represents the key molecule linking cerebrovascular and neuronal function.

Endothelial nitric oxide deficiency promotes Alzheimer's disease pathology

Aging and the presence of cerebrovascular disease are associated with increased incidence of Alzheimer's disease. A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelium derived NO on amyloid precursor protein (APP) related phenotype in late middle aged (LMA) (14-15month) endothelial nitric oxide synthase deficient (eNOS(-/-) ) mice. APP, β-site APP cleaving enzyme (BACE) 1, and amyloid beta (Aβ) levels were significantly higher in the brains of LMA eNOS(-/-) mice as compared with LMA wild-type controls. APP and Aβ1-40 were increased in hippocampal tissue of eNOS(-/-) mice as compared with wild-type mice. LMA eNOS(-/-) mice displayed an increased inflammatory phenotype as compared with LMA wild-type mice. Importantly, LMA eNOS(-/-) mice performed worse in a radial arm maze test of spatial learning and memory as compared with LMA wild-type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline. Cardiovascular risk factors are associated with increased incidence of Alzheimer's disease (AD). A common feature of these risk factors is decreased endothelial nitric oxide (NO). We observed, in mice deficient in endothelial nitric oxide synthase, increased amyloid precursor protein (APP), β-site APP cleaving enzyme 1, amyloid beta levels, microglial activation, and impaired spatial memory. This suggests chronic loss of endothelial NO may be an important contributor to the pathogenesis of sporadic AD.

Knock, Knock: Who’s There?

It has become apparent that major sources of reactive oxygen species (ROS) in mammalian cells are the NADPH oxidases. These multimeric enzymes are composed of membrane-bound catalytic subunits, or Nox enzymes, and a small docking subunit called p22phox. Depending on the Nox isoform, cytosolic regulatory subunits also play a role in their activation. There are 7 Nox isoforms. The first identified was Nox2, also called gp91 phox , which is present in phagocytic cells and is responsible for the oxidative burst. Unlike Nox2, the other Nox enzymes exhibit sustained production of ROS at somewhat lower levels. Their modes of activation differ, as do their cellular distributions. Nox1 through Nox4 are expressed in vascular cells in rodents. Nox5 has been identified in atherosclerotic lesions of humans but is not expressed in rodents. Stimulation of cells with angiotensin II, cytokines, catecholamines, high glucose, or mechanical stretch promotes NADPH oxidase activation, in large part as a result of recruitment of cytosolic subunits to the membrane subunit, leading to the formation of the functional enzyme complex that can transfer electrons to molecular oxygen and the formation of superoxide (O2·−).1 O2·−serves as a progenitor for other ROS, including hydrogen peroxide, peroxynitrite, and hypochlorous acid. Enhanced activity of the NADPH oxidases and increased expression of its subunits occur in many pathological conditions, including hypertension, diabetes mellitus, atherosclerosis, cardiac hypertrophy, and heart failure. Studies of genetically altered mice have shown that the deletion of various NADPH oxidase subunits protects against these diseases and their overexpression promotes pathology.2−4 Article see p 1888 A consequence of excessive ROS formation is loss of endothelial nitric oxide (NO), which can occur by rapid radical-radical reactions with O2·− and other radicals or as a result of dysfunction of the endothelial NO synthase. …

Aged eNOS−/− mice display increased APP expression, microglial activation, and impaired spatial memory

Aging and other cardiovascular risk factors are associated with increased incidence of Alzheimer's disease (AD). A common feature of cardiovascular risk factors is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelial NO on amyloid precursor protein (APP) related phenotype in aged (14–15 month) endothelial nitric oxide synthase deficient (eNOS−/−) mice. APP, β‐site APP cleaving enzyme 1, and amyloid beta (Aβ) levels were significantly higher in the brains of aged eNOS−/− mice as compared to aged wild type mice. APP and Aβ1–40 were increased in hippocampal tissue of eNOS−/− mice as compared to wild type mice. Aged eNOS−/− mice displayed an increased inflammatory phenotype as compared to aged wild type mice. Importantly, aged eNOS−/− mice performed worse in a radial arm maze test of spatial learning and memory as compared to aged wild type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline. Research was supported by NIH grants HL‐111062 and HL‐91867, the Mayo Alzheimer's Disease Research Center (Z.S.K.), AHA scientist development grant (0835436N, A.V.S.), AHA postdoctoral fellowship 12POST8550003 (S.A.A.), Clinical Pharmacology Training Grant (T32 GM08685) (Trainee S.A.A.), the Samuel Johnson Foundation for Genomics of Addiction Program at Mayo Clinic (D.‐S.C.) and the Mayo Foundation.

Abstract 3063: Nitroglycerin Treatment Attenuates APP and BACE1 Protein Levels In Cerebral Microcirculation

Several cardiovascular risk factors are associated with an increased incidence of Alzheimer’s disease (AD); however, the mechanistic link is unknown. A common feature of cardiovascular risk factors is endothelial dysfunction, specifically, loss of bioavailability of endothelial nitric oxide (NO). Recently, we demonstrated that loss of endothelial NO led to increased protein levels of amyloid precursor protein (APP), β-site APP cleaving enzyme 1 (BACE1) and beta amyloid (Aβ) peptide. APP, the parent protein, when sequentially cleaved by BACE1 and γ-secretase generates amyloidogenic, Aβ, the primary component of AD plaques. Our data was the first to demonstrate a possible mechanism linking endothelial dysfunction, via loss of endothelial NO, with the upregulation of important AD related proteins in the cerebral microvasculature. Therefore, we sought to determine if supplementation of cultured human brain microvascular endothelial cells with NO, by utilizing the NO donor, DETA NONOate, would attenuate APP and BACE1 protein. DETA NONOate, 10 -8 to 10 -6 M, reduced expression of APP and BACE1 as compared to untreated control cells (n=3). To test the effects of NO supplementation in vivo, we treated endothelial nitric oxide synthase deficient (eNOS -/- ) mice with 30 mg/kg nitroglycerine b.i.d. or vehicle via subcutaneous injections for 3 days. No difference was observed in blood pressure between animals treated with vehicle (n=7) or nitroglycerine (n=6) (P&gt;0.05) or before and after nitroglycerine treatment (n=6, P&gt;0.05). As expected, cerebral microvessles of eNOS -/- animals treated with nitroglycerine displayed increased levels of cGMP as compared to vehicle (n=7-8; P&lt;0.05). Moreover, APP and BACE1 protein levels were statistically lower in cerebral microvessels from eNOS -/- mice treated with nitroglycerine as compared to vehicle treated (n=6-7, APP, P&lt;0.05; BACE1, P&lt;0.05). These data indicate that supplementation with NO is able to suppress APP and BACE1 levels in the cerebrovascular endothelium. Our findings suggest that preservation of NO/cGMP signaling may be an important therapeutic target in preventing and treating mild cognitive impairment as well as AD.

CCR2-Mediated Antiinflammatory Effects of Endothelial Tetrahydrobiopterin Inhibit Vascular Injury-Induced Accelerated Atherosclerosis

Vascular injury results in loss of endothelial nitric oxide (NO), production of reactive oxygen species (ROS), and the initiation of an inflammatory response. Both NO and ROS modulate inflammation through redox-sensitive pathways. Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) that regulates enzymatic synthesis of either nitric oxide or ROS. We hypothesized that endothelial BH4 is an important regulator of inflammation and vascular remodeling. Endothelium-targeted overexpression of GTP cyclohydrolase 1 (GCH), the rate limiting enzyme in BH4 synthesis, increased levels of tetrahydrobiopterin (BH4), reduced endothelial superoxide, improved eNOS coupling, and reduced vein graft atherosclerosis in transgenic GCH/ApoE-KO mice compared to ApoE-KO controls. Immunohistochemistry using anti-MAC-3 and MAC-1 antibody staining revealed a marked reduction in vein graft macrophage content, as did RT-PCR expression of macrophage marker CD68 mRNA levels in GCH/ApoE-KO mice. When we investigated the potential mediators of this reduction, we discovered that mRNA and protein levels of MCP-1 (CCL2) but not RANTES (CCL5) were significantly reduced in GCH/ApoE-KO aortic tissue. Consistent with this finding we found a decrease in CCR2-mediated, but not CCR5-mediated, chemotaxis in vascular tissue and plasma samples from GCH/ApoE-KO animals. Increased endothelial BH4 reduces vein graft neointimal hyperplasia and atherosclerosis through a reduction in vascular inflammation. These findings highlight the importance of MCP-1/CCR2 signaling in the response to vascular injury and identify novel pathways linking endothelial BH4 to inflammation and vascular remodeling.

Plasma membrane-associated endothelial nitric oxide synthase and activity in aging rat aortic vascular endothelia markedly decline with age

The mechanisms leading to the age-related loss of endothelial nitric oxide (NO) and NO-dependent vasodilation remain largely unknown. Freshly isolated endothelium from young (6 months) and old (36 months) F344 × BrN rats were analyzed for endothelial nitric oxide synthase (eNOS) protein, its subcellular distribution, and association with regulatory proteins. Results show that both vessel ring vasoreactivity and A23187-induced eNOS activity in isolated endothelial cells significantly ( p ⩽ 0.05) declined with age. Levels of cGMP, a reliable marker for NO bioactivity also declined significantly ( p ⩽ 0.01). However, no change in overall eNOS protein was evident. Subcellular fractionation studies revealed an age-related loss in active, plasma membrane-bound eNOS relative to eNOS in the Golgi/cytosol of the endothelium. Plasma membrane-associated eNOS in aged endothelium was also less complexed with the activating proteins Hsp90 and Akt and more associated with to caveolin-1, which inhibits eNOS activity. These results suggest that age-dependent loss of NO may be partly caused by differences in eNOS subcellular distribution and its association with inhibitory proteins.

Angiotensin II causes vascular inflammation via activation of μ‐calpain

Hyperglycemia increases tissue angiotensin II (AngII) levels, which induces endothelial dysfunction and vascular inflammation. We have previously reported that hyperglycemia increases μ-calpain activity causing loss of endothelial nitric oxide and increased leukocyte trafficking in the microcirculation. We report now evidence that links the inflammatory action of AngII to μ-calpain activity. We studied leukocyte-endothelial cell interactions in rats injected with a sub-pressor dose of AngII (23 ng/kg/ip, 18 hrs before study), using intravital microscopy of mesenteric post-capillary venules. AngII increased leukocyte rolling, adhesion and extravasation in the rat mesentery (p<0.01 vs vehicle), in the absence of relevant hemodynamic changes. AngII also increased albumin permeability in the microcirculation, as detected by Texas Red-conjugated albumin (p<0.01). These inflammatory effects of AngII were blocked by administration of the selective calpain inhibitor ZLLal (72 μg/Kg/ip, 18 hrs before study). Western blot analyses of the ubiquitously expressed μ- and m-calpain isoforms in vascular segments of the rat mesentery, demonstrated increased μ-calpain activity in response to AngII. Our data provide a new mechanism for the inflammatory actions of the renin/angiotensin system and further support a role for calpains in diabetic vascular complications. (Supported by NIH Grant # R01DK064344)

Tetrahydrobiopterin: Mediator of Endothelial Protection

During the past twenty years, the essential role of endothelial cells in preservation of vascular homeostasis has been well established.1,2⇓ Protection of the vascular endothelium against harmful influences of circulating substances including excessive levels of lipids has been a major therapeutic approach in prevention of atherosclerosis. While endothelial dysfunction, defined as a loss of biologically active nitric oxide (NO) produced in the endothelium, has been recognized as a prime target for prevention and reversal of atherosclerotic process, identification of exact molecular mechanisms responsible for the loss of endothelial NO have been more difficult to determine. In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Alp et al3 report a series of elegant studies on genetically modified mice supporting the concept that NO synthase cofactor, tetrahydrobiopterin (Figure 1), is an important vascular protective molecule. The beneficial effect of tetrahydrobiopterin in apolipoprotein E (ApoE)-deficient mice appears to be mediated by preservation of endothelial nitric oxide synthase (eNOS) activity and production of nitric oxide in endothelial cells. The major steps of tetrahydrobiopterin biosynthesis. GTP=guanosine 5′-triphosphate. See page 445 In the early 1990s it was recognized that in the presence of suboptimal concentrations of tetrahydrobiopterin, activation of purified neuronal nitric oxide synthase (nNOS) causes “uncoupling of NOS” with subsequent increase …

Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice.

Increased production of reactive oxygen species and loss of endothelial nitric oxide (NO) bioactivity are key features of vascular disease states such as atherosclerosis. Tetrahydrobiopterin (BH4) is a required cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS); pharmacologic studies suggest that reduced BH4 availability may be an important mediator of endothelial dysfunction in atherosclerosis. We aimed to investigate the importance of endothelial BH4 availability in atherosclerosis using a transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GTPCH). Transgenic mice were crossed into an ApoE knockout (ApoE-KO) background and fed a high-fat diet for 16 weeks. Compared with ApoE-KO controls, transgenic mice (ApoE-KO/GCH-Tg) had higher aortic BH4 levels, reduced endothelial superoxide production and eNOS uncoupling, increased cGMP levels, and preserved NO-mediated endothelium dependent vasorelaxations. Furthermore, aortic root atherosclerotic plaque was significantly reduced in ApoE-KO/GCH-Tg mice compared with ApoE-KO controls. These findings indicate that BH4 availability is a critical determinant of eNOS regulation in atherosclerosis and is a rational therapeutic target to restore NO-mediated endothelial function and reduce disease progression.

Effects of sustained low-flow perfusion on the response to vasoconstrictor agents in postnatal intestine.

This laboratory has previously reported that sustained reduction of blood flow in newborn intestine causes a triphasic increase in vascular resistance that occurs over 3-4 h and that these changes are mediated, in part, by loss of endothelial nitric oxide (NO) production. This study examines the effects of exposure to sustained low-flow perfusion on the subsequent response to three contractile agonists: ANG II, norepinephrine (NE), and endothelin-1 (ET-1). Gut loops from 3- and 35-day-old swine were exposed to low-flow conditions in vivo (i.e., reduction of flow to approximately 50% of baseline) for 30 min or 5 h. Thereafter, they were removed to an extracorporeal perfusion circuit for in vitro hemodynamic assessment; alternatively, the mesenteric artery perfusing the gut loop was removed and cut into rings for assessment of isometric tension development. Gut loops from 3-day-old subjects exposed to low-flow conditions demonstrated significantly increased contractile responses to ANG II, NE, and ET-1; also, mesenteric artery rings from these gut loops demonstrated a significant reduction of the ED50 for all three agonists. Similar changes were not observed in intestine or mesenteric artery rings from older subjects. Sustained blockade of endogenous NO synthesis with NG-monomethyl- L-arginine duplicated the effects of exposure to sustained low-flow perfusion. It appears that sustained reduction of blood flow in newborn intestine decreases constitutive NO production, which in turn causes a generalized enhancement of the contractile efficacy of ANG II, NE, and ET-1.