Glutamine Deficiency Promotes Immune and Endothelial Cell Dysfunction in COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has caused the death of almost 7 million people worldwide. While vaccinations and new antiviral drugs have greatly reduced the number of COVID-19 cases, there remains a need for additional therapeutic strategies to combat this deadly disease. Accumulating clinical data have discovered a deficiency of circulating glutamine in patients with COVID-19 that associates with disease severity. Glutamine is a semi-essential amino acid that is metabolized to a plethora of metabolites that serve as central modulators of immune and endothelial cell function. A majority of glutamine is metabolized to glutamate and ammonia by the mitochondrial enzyme glutaminase (GLS). Notably, GLS activity is upregulated in COVID-19, favoring the catabolism of glutamine. This disturbance in glutamine metabolism may provoke immune and endothelial cell dysfunction that contributes to the development of severe infection, inflammation, oxidative stress, vasospasm, and coagulopathy, which leads to vascular occlusion, multi-organ failure, and death. Strategies that restore the plasma concentration of glutamine, its metabolites, and/or its downstream effectors, in conjunction with antiviral drugs, represent a promising therapeutic approach that may restore immune and endothelial cell function and prevent the development of occlusive vascular disease in patients stricken with COVID-19.

Canagliflozin Inhibits Human Endothelial Cell Inflammation through the Induction of Heme Oxygenase-1.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in patients with type 2 diabetes mellitus (T2DM). Studies have also shown that canagliflozin directly acts on endothelial cells (ECs). Since heme oxygenase-1 (HO-1) is an established modulator of EC function, we investigated if canagliflozin regulates the endothelial expression of HO-1, and if this enzyme influences the biological actions of canagliflozin in these cells. Treatment of human ECs with canagliflozin stimulated a concentration- and time-dependent increase in HO-1 that was associated with a significant increase in HO activity. Canagliflozin also evoked a concentration-dependent blockade of EC proliferation, DNA synthesis, and migration that was unaffected by inhibition of HO-1 activity and/or expression. Exposure of ECs to a diabetic environment increased the adhesion of monocytes to ECs, and this was attenuated by canagliflozin. Knockdown of HO-1 reduced the anti-inflammatory effect of canagliflozin which was restored by bilirubin but not carbon monoxide. In conclusion, this study identified canagliflozin as a novel inducer of HO-1 in human ECs. It also found that HO-1-derived bilirubin contributed to the anti-inflammatory action of canagliflozin, but not the anti-proliferative and antimigratory effects of the drug. The ability of canagliflozin to regulate HO-1 expression and EC function may contribute to the clinical profile of the drug.

Targeting Arginine in COVID-19-Induced Immunopathology and Vasculopathy.

Coronavirus disease 2019 (COVID-19) represents a major public health crisis that has caused the death of nearly six million people worldwide. Emerging data have identified a deficiency of circulating arginine in patients with COVID-19. Arginine is a semi-essential amino acid that serves as key regulator of immune and vascular cell function. Arginine is metabolized by nitric oxide (NO) synthase to NO which plays a pivotal role in host defense and vascular health, whereas the catabolism of arginine by arginase to ornithine contributes to immune suppression and vascular disease. Notably, arginase activity is upregulated in COVID-19 patients in a disease-dependent fashion, favoring the production of ornithine and its metabolites from arginine over the synthesis of NO. This rewiring of arginine metabolism in COVID-19 promotes immune and endothelial cell dysfunction, vascular smooth muscle cell proliferation and migration, inflammation, vasoconstriction, thrombosis, and arterial thickening, fibrosis, and stiffening, which can lead to vascular occlusion, muti-organ failure, and death. Strategies that restore the plasma concentration of arginine, inhibit arginase activity, and/or enhance the bioavailability and potency of NO represent promising therapeutic approaches that may preserve immune function and prevent the development of severe vascular disease in patients with COVID-19.

Effects of Sodium-Glucose Co-Transporter 2 Inhibitors on Vascular Cell Function and Arterial Remodeling.

Cardiovascular disease is the leading cause of morbidity and mortality in diabetes. Recent clinical studies indicate that sodium-glucose co-transporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in patients with diabetes. The mechanism underlying the beneficial effect of SGLT2 inhibitors is not completely clear but may involve direct actions on vascular cells. SGLT2 inhibitors increase the bioavailability of endothelium-derived nitric oxide and thereby restore endothelium-dependent vasodilation in diabetes. In addition, SGLT2 inhibitors favorably regulate the proliferation, migration, differentiation, survival, and senescence of endothelial cells (ECs). Moreover, they exert potent antioxidant and anti-inflammatory effects in ECs. SGLT2 inhibitors also inhibit the contraction of vascular smooth muscle cells and block the proliferation and migration of these cells. Furthermore, studies demonstrate that SGLT2 inhibitors prevent postangioplasty restenosis, maladaptive remodeling of the vasculature in pulmonary arterial hypertension, the formation of abdominal aortic aneurysms, and the acceleration of arterial stiffness in diabetes. However, the role of SGLT2 in mediating the vascular actions of these drugs remains to be established as important off-target effects of SGLT2 inhibitors have been identified. Future studies distinguishing drug- versus class-specific effects may optimize the selection of specific SGLT2 inhibitors in patients with distinct cardiovascular pathologies.

Targeting Heme Oxygenase-1 in the Arterial Response to Injury and Disease.

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme into carbon monoxide (CO), iron, and biliverdin, which is rapidly metabolized to bilirubin. The activation of vascular smooth muscle cells (SMCs) plays a critical role in mediating the aberrant arterial response to injury and a number of vascular diseases. Pharmacological induction or gene transfer of HO-1 improves arterial remodeling in animal models of post-angioplasty restenosis, vascular access failure, atherosclerosis, transplant arteriosclerosis, vein grafting, and pulmonary arterial hypertension, whereas genetic loss of HO-1 exacerbates the remodeling response. The vasoprotection evoked by HO-1 is largely ascribed to the generation of CO and/or the bile pigments, biliverdin and bilirubin, which exert potent antioxidant and anti-inflammatory effects. In addition, these molecules inhibit vascular SMC proliferation, migration, apoptosis, and phenotypic switching. Several therapeutic strategies are currently being pursued that may allow for the targeting of HO-1 in arterial remodeling in various pathologies, including the use of gene delivery approaches, the development of novel inducers of the enzyme, and the administration of unique formulations of CO and bilirubin.

Amino Acids in Circulatory Function and Health.

Cardiovascular disease is the major cause of global mortality and disability. Abundant evidence indicates that amino acids play a fundamental role in cardiovascular physiology and pathology. Decades of research established the importance of L-arginine in promoting vascular health through the generation of the gas nitric oxide. More recently, L-glutamine, L-tryptophan, and L-cysteine have also been shown to modulate vascular function via the formation of a myriad of metabolites, including a number of gases (ammonia, carbon monoxide, hydrogen sulfide, and sulfur dioxide). These amino acids and their metabolites preserve vascular homeostasis by regulating critical cellular processes including proliferation, migration, differentiation, apoptosis, contractility, and senescence. Furthermore, they exert potent anti-inflammatory and antioxidant effects in the circulation, and block the accumulation of lipids within the arterial wall. They also mitigate known risk factors for cardiovascular disease, including hypertension, hyperlipidemia, obesity, and diabetes. However, in some instances, the metabolism of these amino acids through discrete pathways yields compounds that fosters vascular disease. While supplementation with amino acid monotherapy targeting the deficiency has ameliorated arterial disease in many animal models, this approach has been less successful in the clinic. A more robust approach combining amino acid supplementation with antioxidants, anti-inflammatory agents, and/or specific amino acid enzymatic pathway inhibitors may prove more successful. Alternatively, supplementation with amino acid-derived metabolites rather than the parent molecule may elicit beneficial effects while bypassing potentially harmful pathways of metabolism. Finally, there is an emerging recognition that circulating levels of multiple amino acids are perturbed in vascular disease and that a more holistic approach that targets all these amino acid derangements is required to restore circulatory function in diseased blood vessels.

Endothelial sodium channel activation promotes cardiac stiffness and diastolic dysfunction in Western diet fed female mice.

OBJECTIVE: Obesity is associated with myocardial fibrosis and impaired diastolic relaxation, abnormalities that are especially prevalent in women. Normal coronary vascular endothelial function is integral in mediating diastolic relaxation, and recent work suggests increased activation of the endothelial cell (EC) mineralocorticoid receptor (ECMR) is associated with impaired diastolic relaxation. As the endothelial Na+ channel (EnNaC) is a downstream target of the ECMR, we sought to determine whether EC-specific deletion of the critical alpha subunit, αEnNaC, would prevent diet induced-impairment of diastolic relaxation in female mice. METHODS AND MATERIALS: Female αEnNaC KO mice and littermate controls were fed a Western diet (WD) high in fat (46%), fructose corn syrup (17.5%) and sucrose (17.5%) for 12-16 weeks. Measurements were conducted for in vivo cardiac function, in vitro cardiomyocyte stiffness and EnNaC activity in primary cultured ECs. Additional biochemical studies examined indicators of oxidative stress, including aspects of antioxidant Nrf2 signaling, in cardiac tissue. RESULTS: Deletion of αEnNaC in female mice fed a WD significantly attenuated WD mediated impairment in diastolic relaxation. Improved cardiac relaxation was accompanied by decreased EnNaC-mediated Na+ currents in ECs and reduced myocardial oxidative stress. Further, deletion of αEnNaC prevented WD-mediated increases in isolated cardiomyocyte stiffness. CONCLUSION: Collectively, these findings support the notion that WD feeding in female mice promotes activation of EnNaC in the vasculature leading to increased cardiomyocyte stiffness and diastolic dysfunction.

Canagliflozin inhibits vascular smooth muscle cell proliferation and migration: Role of heme oxygenase-1.

Recent cardiovascular outcome trials found that sodium-glucose cotransporter-2 (SGLT2) inhibitors reduce cardiovascular disease and mortality in type 2 diabetic patients; however, the underlying mechanisms are not fully known. Since the proliferation and migration of vascular smooth muscle cells (SMCs) contributes to the development of arterial lesions, we hypothesized that SGLT2 inhibitors may exert their beneficial cardiovascular effects by inhibiting the growth and movement of vascular SMCs. Treatment of rat or human aortic SMCs with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation and migration. The inhibition of SMC growth by canagliflozin occurred in the absence of cell death, and was associated with the arrest of SMCs in the G0/G1 phase of the cell cycle and diminished DNA synthesis. Canagliflozin also resulted in the induction of heme oxygenase-1 (HO-1) expression, and a rise in HO activity in vascular SMCs, whereas, empagliflozin or dapagliflozin had no effect on HO activity. Canagliflozin also activated the HO-1 promoter and this was abrogated by mutating the antioxidant responsive element or by overexpressing dominant-negative NF-E2-related factor-2 (Nrf2). The induction of HO-1 by canagliflozin relied on reactive oxygen species (ROS) formation and was negated by antioxidants. Finally, silencing HO-1 expression partially rescued the proliferative and migratory response of canagliflozin-treated SMCs, and this was reversed by carbon monoxide and bilirubin. In conclusion, the present study identifies canagliflozin as a novel inhibitor of vascular SMC proliferation and migration. Moreover, it demonstrates that canagliflozin stimulates the expression of HO-1 in vascular SMCs via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cellular actions of canagliflozin. The ability of canagliflozin to exert these pleiotropic effects may contribute to the favorable clinical actions of the drug and suggest an extra potential benefit of canagliflozin relative to other SGLT2 inhibitors.

The Emerging Role of l-Glutamine in Cardiovascular Health and Disease.

Emerging evidence indicates that l-glutamine (Gln) plays a fundamental role in cardiovascular physiology and pathology. By serving as a substrate for the synthesis of DNA, ATP, proteins, and lipids, Gln drives critical processes in vascular cells, including proliferation, migration, apoptosis, senescence, and extracellular matrix deposition. Furthermore, Gln exerts potent antioxidant and anti-inflammatory effects in the circulation by inducing the expression of heme oxygenase-1, heat shock proteins, and glutathione. Gln also promotes cardiovascular health by serving as an l-arginine precursor to optimize nitric oxide synthesis. Importantly, Gln mitigates numerous risk factors for cardiovascular disease, such as hypertension, hyperlipidemia, glucose intolerance, obesity, and diabetes. Many studies demonstrate that Gln supplementation protects against cardiometabolic disease, ischemia-reperfusion injury, sickle cell disease, cardiac injury by inimical stimuli, and may be beneficial in patients with heart failure. However, excessive shunting of Gln to the Krebs cycle can precipitate aberrant angiogenic responses and the development of pulmonary arterial hypertension. In these instances, therapeutic targeting of the enzymes involved in glutaminolysis such as glutaminase-1, Gln synthetase, glutamate dehydrogenase, and amino acid transaminase has shown promise in preclinical models. Future translation studies employing Gln delivery approaches and/or glutaminolysis inhibitors will determine the success of targeting Gln in cardiovascular disease.

Canagliflozin Inhibits Human Endothelial Cell Proliferation and Tube Formation.

Recent clinical trials revealed that sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly reduce cardiovascular events in type 2 diabetic patients, however, canagliflozin increased limb amputations, an effect not seen with other SGLT2 inhibitors. Since endothelial cell (EC) dysfunction promotes diabetes-associated vascular disease and limb ischemia, we hypothesized that canagliflozin, but not other SGLT2 inhibitors, impairs EC proliferation, migration, and angiogenesis. Treatment of human umbilical vein ECs (HUVECs) with clinically relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin, inhibited cell proliferation. In particular, 10 µM canagliflozin reduced EC proliferation by approximately 45%. The inhibition of EC growth by canagliflozin occurred in the absence of cell death and was associated with diminished DNA synthesis, cell cycle arrest, and a striking decrease in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer partially rescued the proliferative response of HUVECs treated with canagliflozin. A high concentration of canagliflozin (50 µM) modestly inhibited HUVEC migration by 20%, but markedly attenuated their tube formation by 65% and EC sprouting from mouse aortas by 80%. A moderate 20% reduction in HUVEC migration was also observed with a high concentration of empagliflozin (50 µM), while neither empagliflozin nor dapagliflozin affected tube formation by HUVECs. The present study identified canagliflozin as a robust inhibitor of human EC proliferation and tube formation. The anti-proliferative action of canagliflozin occurs in the absence of cell death and is due, in part, to the blockade of cyclin A expression. Notably, these actions are not seen with empagliflozin or dapagliflozin. The ability of canagliflozin to exert these pleiotropic effects on ECs may contribute to the clinical actions of this drug.

Glutaminase-1 stimulates the proliferation, migration, and survival of human endothelial cells.

Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G0/G1 phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.

Arginase inhibition prevents the development of hypertension and improves insulin resistance in obese rats.

This study investigated the temporal activation of arginase in obese Zucker rats (ZR) and determined if arginase inhibition prevents the development of hypertension and improves insulin resistance in these animals. Arginase activity, plasma arginine and nitric oxide (NO) concentration, blood pressure, and insulin resistance were measured in lean and obese animals. There was a chronological increase in vascular and plasma arginase activity in obese ZR beginning at 8 weeks of age. The increase in arginase activity in obese animals was associated with a decrease in insulin sensitivity and circulating levels of arginine and NO. The rise in arginase activity also preceded the increase in blood pressure in obese ZR detected at 12 weeks of age. Chronic treatment of 8-week-old obese animals with an arginase inhibitor or L-arginine for 4 weeks prevented the development of hypertension and improved plasma concentrations of arginine and NO. Arginase inhibition also improved insulin sensitivity in obese ZR while L-arginine supplementation had no effect. In conclusion, arginase inhibition prevents the development of hypertension and improves insulin sensitivity while L-arginine administration only mitigates hypertension in obese animals. Arginase represents a promising therapeutic target in ameliorating obesity-associated vascular and metabolic dysfunction.

Preconditioning with the BKCa channel activator NS-1619 prevents ischemia-reperfusion-induced inflammation and mucosal barrier dysfunction: roles for ROS and heme oxygenase-1.

Activation of large-conductance Ca2+-activated K+ (BKCa) channels evokes cell survival programs that mitigate intestinal ischemia and reperfusion (I/R) inflammation and injury 24 h later. The goal of the present study was to determine the roles of reactive oxygen species (ROS) and heme oxygenase (HO)-1 in delayed acquisition of tolerance to I/R induced by pretreatment with the BKCa channel opener NS-1619. Superior mesentery arteries were occluded for 45 min followed by reperfusion for 70 min in wild-type (WT) or HO-1-null (HO-1-/-) mice that were pretreated with NS-1619 or saline vehicle 24 h earlier. Intravital microscopy was used to quantify the numbers of rolling and adherent leukocytes. Mucosal permeability, tumor necrosis factor-α (TNF-α) levels, and HO-1 activity and expression in jejunum were also determined. I/R induced leukocyte rolling and adhesion, increased intestinal TNF-α levels, and enhanced mucosal permeability in WT mice, effects that were largely abolished by pretreatment with NS-1619. The anti-inflammatory and mucosal permeability-sparing effects of NS-1619 were prevented by coincident treatment with the HO-1 inhibitor tin protoporphyrin-IX or a cell-permeant SOD mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), in WT mice. NS-1619 also increased jejunal HO-1 activity in WT animals, an effect that was attenuated by treatment with the BKCa channel antagonist paxilline or MnTBAP. I/R also increased postischemic leukocyte rolling and adhesion and intestinal TNF-α levels in HO-1-/- mice to levels comparable to those noted in WT animals. However, NS-1619 was ineffective in preventing these effects in HO-1-deficient mice. In summary, our data indicate that NS-1619 induces the development of an anti-inflammatory phenotype and mitigates postischemic mucosal barrier disruption in the small intestine by a mechanism that may involve ROS-dependent HO-1 activity.NEW & NOTEWORTHY Antecedent treatment with the large-conductance Ca2+-activated K+ channel opener NS-1619 24 h before ischemia-reperfusion limits postischemic tissue injury by an oxidant-dependent mechanism. The present study shows that NS-1619-induced oxidant production prevents ischemia-reperfusion-induced inflammation and mucosal barrier disruption in the small intestine by provoking increases in heme oxygenase-1 activity.

Daily exercise prevents diastolic dysfunction and oxidative stress in a female mouse model of western diet induced obesity by maintaining cardiac heme oxygenase-1 levels.

OBJECTIVE: Obesity is a global epidemic with profound cardiovascular disease (CVD) complications. Obese women are particularly vulnerable to CVD, suffering higher rates of CVD compared to non-obese females. Diastolic dysfunction is the earliest manifestation of CVD in obese women but remains poorly understood with no evidence-based therapies. We have shown early diastolic dysfunction in obesity is associated with oxidative stress and myocardial fibrosis. Recent evidence suggests exercise may increase levels of the antioxidant heme oxygenase-1 (HO-1). Accordingly, we hypothesized that diastolic dysfunction in female mice consuming a western diet (WD) could be prevented by daily volitional exercise with reductions in oxidative stress, myocardial fibrosis and maintenance of myocardial HO-1 levels. MATERIALS/METHODS: Four-week-old female C57BL/6J mice were fed a high-fat/high-fructose WD for 16weeks (N=8) alongside control diet fed mice (N=8). A separate cohort of WD fed females was allowed a running wheel for the entire study (N=7). Cardiac function was assessed at 20weeks by high-resolution cardiac magnetic resonance imaging (MRI). Functional assessment was followed by immunohistochemistry, transmission electron microscopy (TEM) and Western blotting to identify pathologic mechanisms and assess HO-1 protein levels. RESULTS: There was no significant body weight decrease in exercising mice, normalized body weight 14.3g/mm, compared to sedentary mice, normalized body weight 13.6g/mm (p=0.38). Total body fat was also unchanged in exercising, fat mass of 6.6g, compared to sedentary mice, fat mass 7.4g (p=0.55). Exercise prevented diastolic dysfunction with a significant reduction in left ventricular relaxation time to 23.8ms for exercising group compared to 33.0ms in sedentary group (p<0.01). Exercise markedly reduced oxidative stress and myocardial fibrosis with improved mitochondrial architecture. HO-1 protein levels were increased in the hearts of exercising mice compared to sedentary WD fed females. CONCLUSIONS: This study provides seminal evidence that exercise can prevent diastolic dysfunction in WD-induced obesity in females even without changes in body weight. Furthermore, the reduction in myocardial oxidative stress and fibrosis and improved HO-1 levels in exercising mice suggests a novel mechanism for the antioxidant effect of exercise.

Ammonia promotes endothelial cell survival via the heme oxygenase-1-mediated release of carbon monoxide.

Although endothelial cells produce substantial quantities of ammonia during cell metabolism, the physiologic role of this gas in these cells is not known. In this study, we investigated if ammonia regulates the expression of heme oxygenase-1 (HO-1), and if this enzyme influences the biological actions of ammonia on endothelial cells. Exogenously administered ammonia, given as ammonium chloride or ammonium hydroxide, or endogenously generated ammonia stimulated HO-1 protein expression in cultured human and murine endothelial cells. Dietary supplementation of ammonia also induced HO-1 protein expression in murine arteries. The increase in HO-1 protein by ammonia in endothelial cells was first detected 4h after ammonia exposure and was associated with the induction of HO-1 mRNA, enhanced production of reactive oxygen species (ROS), and increased expression and activity of NF-E2-related factor-2 (Nrf2). Ammonia also activated the HO-1 promoter and this was blocked by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. The induction of HO-1 expression by ammonia was dependent on ROS formation and prevented by N-acetylcysteine or rotenone. Finally, prior treatment of endothelial cells with ammonia inhibited tumor necrosis factor-α-stimulated cell death. However, silencing HO-1 expression abrogated the protective action of ammonia and this was reversed by the administration of carbon monoxide but not bilirubin or iron. In conclusion, this study demonstrates that ammonia stimulates the expression of HO-1 in endothelial cells via the ROS-Nrf2 pathway, and that the induction of HO-1 contributes to the cytoprotective action of ammonia by generating carbon monoxide. Moreover, it identifies ammonia as a potentially important signaling gas in the vasculature that promotes endothelial cell survival.

Heme oxygenase-1-derived bilirubin counteracts HIV protease inhibitor-mediated endothelial cell dysfunction.

The use of HIV protease inhibitors (PIs) has extended the duration and quality of life for HIV-positive individuals. However there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we investigated the effect of HIV PIs on EC function and determined if the enzyme heme oxygenase (HO-1) influences the biological action of these drugs. We found that three distinct PIs, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). PIs also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the PI-mediated induction of HO-1 was abolished by N-acetyl-l-cysteine and rotenone. Furthermore, PIs blocked EC proliferation and migration and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition of HO-1 activity or expression potentiated the anti-proliferative and inflammatory actions of PIs which was reversed by bilirubin but not carbon monoxide. Alternatively, adenovirus-mediated overexpression of HO-1 attenuated the growth-inhibitory and inflammatory effect of PIs. In contrast, blocking HO-1 activity failed to modify the anti-migratory effect of the PIs. Thus, induction of HO-1 via the ROS-Nrf2 pathway in human ECs counteracts the anti-proliferative and inflammatory actions of PIs by generating bilirubin. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing EC dysfunction and vascular disease in HIV-infected patients undergoing antiretroviral therapy.

Prolonged cyclic strain inhibits human endothelial cell growth.

The vascular endothelium is continuously exposed to cyclic mechanical strain due to the periodic change in vessel diameter as a result of pulsatile blood flow. Since emerging evidence indicates the cyclic strain plays an integral role in regulating endothelial cell function, the present study determined whether application of a physiologic regimen of cyclic strain (6% at 1 hertz) influences the proliferation of human arterial endothelial cells. Prolonged exposure of human dermal microvascular or human aortic endothelial cells to cyclic strain for up to 7 days resulted in a marked decrease in cell growth. The strain-mediated anti-proliferative effect was associated with the arrest of endothelial cells in the G2/M phase of the cell cycle, did not involve cell detachment or cytotoxicity, and was due to the induction of p21. Interestingly, the inhibition in endothelial cell growth was independent of the strain regimen since prolonged application of constant or intermittent 6% strain was also able to block endothelial cell proliferation. The ability of chronic physiologic cyclic strain to inhibit endothelial cell growth represents a previously unrecognized mechanism by which hemodynamic forces maintain these cells in a quiescent, non-proliferative state.