Abstract 12185: Restoration of Autophagy in Endothelial Cells From Patients With Diabetes Improves Nitric Oxide Signaling

Diabetes is associated with oxidative stress and decreased nitric oxide bioactivity in the vasculature. Autophagy is a critical multistep pathway that eliminates damaged proteins and organelles from the cell. Emerging evidence suggests impaired autophagy in non-vascular tissues contributes to the pathogenesis of diabetes. We hypothesized that impaired autophagy contributes to endothelial dysfunction associated with diabetes in humans. We measured vascular function and autophagy markers in freshly isolated endothelial cells (ECs) from patients with diabetes and non-diabetic controls. Diabetes was associated with endothelial dysfunction characterized by lower brachial artery flow-mediated dilation and impaired activation of eNOS by insulin in ECs. ECs from diabetic patients had higher levels of p62 (Figure), a protein that accumulates with reduced autophagic flux. Measures of autophagy initiation including rapamycin stimulation, beclin 1 levels, and LC3 puncta were not different. Global activation of autophagy with spermidine reversed endothelial dysfunction in freshly isolated ECs from diabetic patients (Figure). In ECs from controls, inhibiting autophagy with bafilomycin impaired eNOS activation confirming that intact autophagy promotes NO signaling. Evidence from cultured endothelial cells in high glucose conditions suggested that lysosomal function is intact as measured by lysosomal number and acidification. Blocking the terminal step of autophagy with bafilomycin in ECs from diabetics led to a further accumulation of p62, suggesting intact but insufficient levels of autophagy. Lamp2a, which facilitates the merger of autophagosomes and lysosomes, was higher in diabetic cells, possibly reflecting a compensatory response to reduced flux. These findings provide evidence for inadequate autophagic flux in ECs from diabetic patients that contributes to impaired NO signaling and may be a target for therapy of diabetic vascular disease.

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Abstract 516: Altered Autophagy Contributes to Endothelial Dysfunction in Human Diabetes Mellitus

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.516 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Jessica L Fetterman ◽

Nir Flint ◽

Monica Holbrook ◽

Erika A Linder ◽

Brittany D Berk ◽

...

Keyword(s):

Diabetes Mellitus ◽

Endothelial Cells ◽

Endothelial Dysfunction ◽

Vascular Function ◽

Experimental Models ◽

Diabetic Patients ◽

Autophagic Flux ◽

Compensatory Response ◽

Patients With Diabetes ◽

Lc3 Puncta

Diabetes is associated with increased oxidative stress and decreased nitric oxide (NO) bioactivity in the vasculature. Autophagy is a critical multistep pathway that eliminates oxidatively damaged proteins and organelles from the cell. Emerging evidence implicates impaired autophagy in a variety of non-vascular tissues that contributes to the pathogenesis of diabetes in experimental models. We hypothesized that impaired autophagy contributes to endothelial dysfunction associated with type 2 diabetes mellitus in humans. We measured vascular function and markers of autophagy in freshly isolated endothelial cells from patients with diabetes (n=41) and non-diabetic controls (n=41). Diabetes was associated with endothelial dysfunction characterized by lower brachial artery flow-mediated dilation. Endothelial cells from diabetic patients displayed no differences in LC3 puncta compared to those from controls but had higher levels of endothelial p62 (p=0.004), a protein that accumulates with reduced autophagic flux. In endothelial cells from controls, the autophagy inhibitor bafilomycin impaired eNOS activation, confirming that intact autophagy is necessary for NO signaling. Global activation of autophagy with spermidine reversed endothelial dysfunction in cultured endothelial cells exposed to high glucose (p=0.007). In regard to mechanism of impairment, the autophagy initiator rapamycin failed to improve eNOS activation in endothelial cells from diabetics. Further, expression of beclin-1, a key contributor to autophagosome formation, was similar in diabetics and controls, arguing against impaired initiation of autophagy. In endothelial cells from diabetics, blocking the terminal step of autophagy with bafilomycin led to a further accumulation of p62 (P=0.01), suggesting intact but insufficient levels of autophagy. Lamp2a, which facilitates the merger of autophagosomes and lysosomes, was higher in diabetic cells, possibly reflecting a compensatory response to reduced flux. Collectively, these findings provide evidence for inadequate autophagic flux in endothelial cells from diabetic patients that contributes to endothelial dysfunction and may be a target for therapy of diabetic vascular disease.

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Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00247.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. L46-L56 ◽

Cited By ~ 51

Author(s):

Shruti Sharma ◽

Neetu Sud ◽

Dean A. Wiseman ◽

A. Lee Carter ◽

Sanjiv Kumar ◽

...

Keyword(s):

Nitric Oxide ◽

Blood Flow ◽

Pulmonary Hypertension ◽

Endothelial Dysfunction ◽

Mitochondrial Dysfunction ◽

Pulmonary Blood Flow ◽

Heat Shock Protein 90 ◽

Nitric Oxide Signaling ◽

Carnitine Metabolism ◽

No Signaling

Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression ( P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs ( P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.

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Abstract 18673: JNK Inhibition Restores Endothelial Function in Type 2 Diabetes Mellitus

Circulation ◽

10.1161/circ.132.suppl_3.18673 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Rosa Breton-Romero ◽

Bihua Feng ◽

Monika Holbrook ◽

Melissa G Farb ◽

Jessica L Fetterman ◽

...

Keyword(s):

Nitric Oxide ◽

Diabetes Mellitus ◽

Insulin Resistance ◽

Type 2 Diabetes ◽

Endothelial Cells ◽

Cardiovascular Diseases ◽

Endothelial Dysfunction ◽

Diabetic Patients ◽

Jnk Activation

Introduction: Diabetes mellitus type 2 is an increasingly public health problem and it is a major cause in the development of cardiovascular diseases. Endothelial dysfunction is a key mechanism that contributes to the pathogenesis of cardiovascular diseases and is a well-known feature of clinical diabetes. Prior studies have demonstrated an impaired nitric oxide bioavailability and a reduced endothelium-dependent vasodilation under diabetic conditions and in animal models, JNK activity has been widely described to be involved in systemic insulin resistance. Hypothesis: Our study aimed to evaluate the involvement of JNK in endothelial dysfunction, studying its potential role in altered eNOS activation and NO synthesis in diabetic patients. Methods: We measured endothelial function and JNK activity in freshly isolated endothelial cells from diabetic patients (n=38) and nondiabetic controls (n=40). Results: ECs from diabetic patients displayed impaired eNOS activation and reduced NO release after insulin and A23187 stimulation, consistent with the presence of endothelial dysfunction. JNK activation was higher in diabetic (**P=0.003), and was associated with lower flow-mediated dilation (r=-0.53, *P=0.02). In endothelial cells from diabetic patients, treatment with JNK chemical inhibitor (SP600125) restored eNOS activation and insulin response (***P<0.001). Nitric oxide bioactivity after A23187 stimuli with diabetes was also recovered in endothelial cells from patients with diabetes. Conclusions: In summary, our data suggest that JNK activation contributes to vascular insulin resistance and endothelial dysfunction in patients with type 2 diabetes and may represent a target in novel therapeutic opportunities.

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The interaction between endothelin-1 and nitric oxide in the vasculature: new perspectives

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00397.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. R1288-R1295 ◽

Cited By ~ 107

Author(s):

Stephane L. Bourque ◽

Sandra T. Davidge ◽

Michael A. Adams

Keyword(s):

Nitric Oxide ◽

Endothelial Dysfunction ◽

Vascular Disease ◽

Vascular Remodeling ◽

Vascular Function ◽

No Synthase ◽

Endothelin 1 ◽

No Signaling ◽

No Bioavailability

Nitric oxide (NO) and endothelin-1 (ET-1) are natural counterparts in vascular function, and it is becoming increasingly clear that an imbalance between these two mediators is a characteristic of endothelial dysfunction and is important in the progression of vascular disease. Here, we review classical and more recent data that suggest that ET-1 should be regarded as an essential component of NO signaling. In particular, we review evidence of the role of ET-1 in models of acute and chronic NO synthase blockade. Furthermore, we discuss the possible mechanisms by which NO modulates ET-1 activity. On the basis of these studies, we suggest that NO tonically inhibits ET-1 function, and in conditions of diminished NO bioavailability, the deleterious effects of unmitigated ET-1 actions result in vasoconstriction and eventually lead to vascular remodeling and dysfunction.

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Abstract P060: High Salt Induces An Endothelial HDAC1-stimulating Circulating Factor Leading To Disrupted Renal Microvascular Nitric Oxide Signaling

Hypertension ◽

10.1161/hyp.76.suppl_1.p060 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Luke S Dunaway ◽

Anthony K Cook ◽

Edward W Inscho ◽

Jennifer Pollock

Keyword(s):

Nitric Oxide ◽

Endothelial Dysfunction ◽

Specific Inhibitor ◽

High Salt ◽

Afferent Arteriole ◽

Sprague Dawley ◽

Salt Diet ◽

Nitric Oxide Signaling ◽

No Signaling

High salt diet (HS) decreases endothelial nitric oxide (NO) signaling as described in human and rodent studies. We have previously shown that inhibition of HDAC1 restores NO signaling in the afferent arteriole of HS fed rats. It remains unknown, however, how HS initiates endothelial dysfunction and activates HDAC1. We first investigated if HS induced endothelial dysfunction is acutely regulated by a circulating factor. To test this, male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or 2 weeks of HS (4.0% NaCl). Afferent arteriole vasoconstrictor responses to the NOS inhibitor, L-NAME, were monitored using the in vitro, blood perfused juxtamedullary nephron preparation. Perfusing arterioles from NS fed rats with HS blood blunted constriction to L-NAME (88 ± 1% of Con) compared to perfusing with NS blood (76 ± 2% of Con; P=0.0003), but not completely as seen in arterioles from HS fed rats perfused with HS blood (96 ± 2% of Con; P=0.0095). HS arterioles perfused with NS blood had similar L-NAME-induced constriction (75 ± 2% of Con) compared to arterioles from NS fed rats perfused with NS blood (P=0.9107). These results suggest HS induces endothelial function through an acute-acting circulating factor. This was not due to increased plasma arginase activity (NS: 7.96 ± 3.73 U/L vs HS: 5.42 ± 1.83 U/L P=0.5813) which competes with NO synthase 3 for arginine, nor was it due to decreased superoxide scavenging capacity of the plasma (NS: 7.96 ± 3.73 U/L vs HS: 5.42 ± 1.83 U/L P=0.5813) as measured by a cytochrome c reduction based assay. We then investigated if HS increased endothelial HDAC1 activity. Renal endothelial cells were isolated via magnetic activated cell sorting from NS and HS fed rats and incubated in plasma from the same rat. HDAC1 activity was monitored as the MS-275 (HDAC1 specific inhibitor) inhibitable portion of total HDAC activity. We found HS significantly increased renal endothelium HDAC1 activity (NS: 0.38 ± 0.03 pmol/min vs HS:0.94 ± 0.19 pmol/min, P=0.01). We conclude that HS disruption of renal microvascular NO signaling is initiated by a circulating factor(s) that is dependent upon increased endothelial HDAC1 activity.

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Glucose scavenging of nitric oxide

AJP Renal Physiology ◽

10.1152/ajprenal.2001.280.3.f480 ◽

2001 ◽

Vol 280 (3) ◽

pp. F480-F486 ◽

Cited By ~ 75

Author(s):

Sergey V. Brodsky ◽

Albert Marcus Morrishow ◽

Nimish Dharia ◽

Steven S. Gross ◽

Michael S. Goligorsky

Keyword(s):

Nitric Oxide ◽

Endothelial Dysfunction ◽

Sharp Decrease ◽

Negative Ion ◽

Diabetic Patients ◽

Chemical Inactivation ◽

Patients With Diabetes ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Endothelial dysfunction accompanies suboptimal glucose control in patients with diabetes mellitus. A hallmark of endothelial dysfunction is a deficiency in production or bioavailability of vascular nitric oxide (NO). Here we demonstrate that acute exposure of human endothelial cells to glucose, at levels found in plasma of diabetic patients, results in a significant blunting of NO responses to the endothelial nitric oxide synthase (eNOS) agonists bradykinin and A-23187. Monitoring of NO generation by purified recombinant bovine eNOS in vitro, using amperometric electrochemical detection and an NO-selective porphyrinic microelectrode, showed that glucose causes a progressive and concentration-dependent attenuation of detectable NO. Addition of glucose to pure NO solutions similarly elicited a sharp decrease in NO concentration, indicating that glucose promotes NO loss. Electrospray ionization-tandem mass spectrometry, using negative ion monitoring, directly demonstrated the occurrence of a covalent reaction involving unitary addition of NO (or a derived species) to glucose. Collectively, our findings reveal that hyperglycemia promotes the chemical inactivation of NO; this glucose-mediated NO loss may directly contribute to hypertension and endothelial dysfunction in diabetic patients.

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Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling

Atherosclerosis ◽

10.1016/j.atherosclerosis.2016.01.043 ◽

2016 ◽

Vol 247 ◽

pp. 207-217 ◽

Cited By ~ 46

Author(s):

Jessica L. Fetterman ◽

Monica Holbrook ◽

Nir Flint ◽

Bihua Feng ◽

Rosa Bretόn-Romero ◽

...

Keyword(s):

Nitric Oxide ◽

Diabetes Mellitus ◽

Endothelial Cells ◽

Nitric Oxide Signaling ◽

Patients With Diabetes

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Crocodile blood supplementation protects vascular function in diabetic mice

Food Production, Processing and Nutrition ◽

10.1186/s43014-021-00066-w ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Chui Yiu Bamboo Chook ◽

Francis M. Chen ◽

Gary Tse ◽

Fung Ping Leung ◽

Wing Tak Wong

Keyword(s):

Endothelial Cells ◽

Vascular Function ◽

Quantitative Polymerase Chain Reaction ◽

Functional Study ◽

Diabetic Patients ◽

Diabetic Mice ◽

Gene Expressions ◽

Inflammatory State ◽

Crocodile Blood

Abstract Cardiovascular disease is a major cause of mortality in diabetic patients due to the heightened oxidative stress and pro-inflammatory state in vascular tissues. Effective approaches targeting cardiovascular health for diabetic patients are urgently needed. Crocodile blood, an emerging dietary supplement, was suggested to have anti-oxidative and anti-inflammatory effects in vitro, which have yet to be proven in animal models. This study thereby aimed to evaluate whether crocodile blood can protect vascular function in diabetic mice against oxidation and inflammation. Diabetic db/db mice and their counterparts db/m+ mice were treated daily with crocodile blood soluble fraction (CBSF) or vehicle via oral gavage for 4 weeks before their aortae were harvested for endothelium-dependent relaxation (EDR) quantification using wire myograph, which is a well-established functional study for vascular function indication. Organ culture experiments culturing mouse aortae from C57BL/6 J mice with or without IL-1β and CBSF were done to evaluate the direct effect of CBSF on endothelial function. Reactive oxygen species (ROS) levels in mouse aortae were assessed by dihydroethidium (DHE) staining with inflammatory markers in endothelial cells quantified by quantitative polymerase chain reaction (qPCR). CBSF significantly improved deteriorated EDR in db/db diabetic mice through both diet supplementation and direct culture, with suppression of ROS level in mouse aortae. CBSF also maintained EDR and reduced ROS levels in mouse aortae against the presence of pro-inflammatory IL-1β. Under the pro-inflammatory state induced by IL-1β, gene expressions of inflammatory cytokines were downregulated, while the protective transcripts UCP2 and SIRT6 were upregulated in endothelial cells. Our study suggests a novel beneficial effect of crocodile blood on vascular function in diabetic mice and that supplementation of diet with crocodile blood may act as a complementary approach to protect against vascular diseases through anti-oxidation and anti-inflammation in diabetic patients. Graphical abstract

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Endothelial dysfunction and body mass index: is there a role for plasma peroxynitrite?

Beni-Suef University Journal of Basic and Applied Sciences ◽

10.1186/s43088-020-00092-6 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Theresa Chikopela ◽

Douglas C. Heimburger ◽

Longa Kaluba ◽

Pharaoh Hamambulu ◽

Newton Simfukwe ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Endothelial Dysfunction ◽

Body Mass ◽

Vascular Function ◽

Aortic Ring ◽

Normal Weight ◽

Oxide Synthase ◽

Weight Groups

Abstract Background Endothelial function is dependent on the balance between vasoconstrictive and vasodilatory substances. The endothelium ability to produce nitric oxide is one of the most crucial mechanisms in regulating vascular tone. An increase in inducible nitric oxide synthase contributes to endothelial dysfunction in overweight persons, while oxidative stress contributes to the conversion of nitric oxide to peroxynitrite (measured as nitrotyrosine in vivo) in underweight persons. The objective of this study was to elucidate the interaction of body composition and oxidative stress on vascular function and peroxynitrite. This was done through an experimental design with three weight groups (underweight, normal weight and overweight), with four treatment arms in each. Plasma nitrotyrosine levels were measured 15–20 h post lipopolysaccharide (LPS) treatment, as were aortic ring tension changes. Acetylcholine (ACh) and sodium nitroprusside (SNP) challenges were used to observe endothelial-dependent and endothelial-independent vascular relaxation after pre-constriction of aortic rings with phenylephrine. Results Nitrotyrosine levels in saline-treated rats were similar among the weight groups. There was a significant increase in nitrotyrosine levels between saline-treated rats and those treated with the highest lipopolysaccharide doses in each of the weight groups. In response to ACh challenge, Rmax (percentage reduction in aortic tension) was lowest in overweight rats (112%). In response to SNP, there was an insignificantly lower Rmax in the underweight rats (106%) compared to the normal weight rats (112%). Overweight rats had a significant decrease in Rmax (83%) in response to SNP, signifying involvement of a more chronic process in tension reduction changes. A lower Rmax accompanied an increase in peroxynitrite after acetylcholine challenge in all weight groups. Conclusions Endothelial dysfunction, observed as an impairment in the ability to reduce tension, is associated with increased plasma peroxynitrite levels across the spectrum of body mass. In higher-BMI rats, an additional role is played by vascular smooth muscle in the causation of endothelial dysfunction.

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