Heme oxygenase-2 protects against ischemic acute kidney injury: influence of age and sex.

Heme oxygenase (HO) activity is exhibited by inducible (HO-1) and constitutive (HO-2) proteins. HO-1 protects against ischemic and nephrotoxic acute kidney injury (AKI). We have previously demonstrated that HO-2 protects against heme protein-induced AKI. The present study examined whether HO-2 is protective in ischemic AKI. Renal ischemia was imposed on young and aged HO-2+/+ and HO-2-/- mice. On days 1 and 2 after renal ischemia, there were no significant differences in renal function between young male HO-2+/+ and HO-2-/- mice, between young female HO-2+/+ and HO-2-/- mice, or between aged female HO-2+/+ and HO-2-/- mice. However, in aged male mice, HO-2 deficiency worsened renal function on days 1 and 2 after ischemic AKI, and, on day 2 after ischemia, such deficiency augmented upregulation of injury-related genes and worsened histological injury. Renal HO activity was markedly decreased in unstressed aged male HO-2-/- mice and remained so after ischemia, despite exaggerated HO-1 induction in HO-2-/- mice after ischemia. Such exacerbation of deficiency of HO-2 protein and HO activity may reflect phosphorylated STAT3, as activation of this proinflammatory transcription factor was accentuated early after ischemia in aged male HO-2-/- mice. This exacerbation may not reflect impaired induction of nephroprotectant genes, since the induction of HO-1, sirtuin 1, and ß-catenin was accentuated in aged male HO-2-/- mice after ischemia. We conclude that aged male mice are hypersensitive to ischemic AKI and that HO-2 mitigates such sensitivity. We speculate that this protective effect of HO-2 may be mediated, at least in part, by suppression of phosphorylated STAT3-dependent signaling.

Role of TLR4 signaling in the nephrotoxicity of heme and heme proteins.

Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the Toll-like receptor-4 (TLR4) receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-κB and its downstream gene monocyte chemoattractant protein-1(MCP-1); in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor Nrf2 and its downstream gene heme oxygenase-1 (HO-1). TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histological injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4+/+ and TLR4-/- mice. We conclude that 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; and 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in virtually all other AKI models studied to date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what occurs in vivo.

The murine dialysis fistula model exhibits a senescence phenotype: pathobiological mechanisms and therapeutic potential.

There is no therapy that promotes maturation and functionality of a dialysis arteriovenous fistula (AVF). The search for such therapies largely relies on evaluation of vascular responses and putative therapies in experimental AVFs. We studied an AVF in mice with chronic kidney disease (CKD). We demonstrate numerous stressors in the vein of the AVF-CKD group, including pathological shear, mitogenic, inflammatory, and hypoxia-reoxygenation stress. Because stress promotes premature senescence, we examined whether senescence is induced in the vein of the AVF-CKD model. We demonstrate a senescence phenotype in the AVF-CKD model, as indicated by increased expression of p16Ink4a, p21Cip1, and p53 and expected changes for certain senescence-associated microRNAs. RNA-sequencing analysis demonstrated differential expression of ~10,000 genes, including upregulation of proinflammatory and proliferative genes, in the vein of the AVF-CKD group. The vein in the AVF-CKD group exhibited telomere erosion and increased senescence-associated ß-galactosidase activity and staining. Senescence was induced in the artery of the AVF-CKD group and in the vein of the AVF without CKD. Finally, given the rapidly rising clinical interest in senolytics, we provide proof of concept of senolytics as a therapeutic approach by demonstrating that senolytics decrease p16Ink4a expression in the AVF-CKD model. This study introduces a novel concept underlying the basis for maturational and functional failure in human dialysis AVFs and identifies a new target for senolytic therapy.

The Role of Interleukin-10 in the Pathophysiology of Preeclampsia.

PURPOSE OF REVIEW: The pathophysiology of preeclampsia is complex and not entirely understood. A key feature in preeclampsia development is an immunological imbalance that shifts the maternal immune response from one of tolerance towards one promoting chronic inflammation and endothelial dysfunction. As a key regulator of immunity, IL-10 not only has immunomodulatory activity, but also directly benefits vasculature and promotes successful cellular interactions at the maternal-fetal interface. Here we focus on the mechanisms by which the dysregulation of IL-10 may contribute to the pathophysiology of preeclampsia. RECENT FINDINGS: Dysregulation of IL-10 has been demonstrated in various animal models of preeclampsia. Decreased IL-10 production in both placenta and peripheral blood mononuclear cells has been reported in human studies, but with inconsistent results. The significance of IL-10 in preeclampsia has shifted from a key biomarker to one with therapeutic potential. As such, a better understanding of the role of this cytokine in the pathophysiology of preeclampsia is of paramount importance.

The Occurrence of Senescence in the Arteriovenous Fistula in the Rat.

BACKGROUND: Maturational failure of dialysis arteriovenous fistulas (AVFs) not uncommonly occurs and is of considerable and timely importance. Our prior studies demonstrate that senescence, a phenotypic process that promotes vascular and other diseases, occurs in the murine AVF. In the present study, we examined whether senescence also occurs in the rat AVF model and the effect of compounds that inhibit or accelerate senescence. METHODS: The rat AVF was created in the femoral vessels by an end vein-side artery anastomosis. We assessed in the AVF the expression of critical drivers of senescence, specifically, the cell cycle inhibitors p16Ink4a and p21Cip1, and such indices of a senescence phenotype as senescence-associated ß-galactosidase (SA-ß-gal) activity, SA-ß-gal staining, and a senescence-associated secretory phenotype (SASP). We examined the effects of compounds that retard or accelerate senescence on AVF blood flow. RESULTS: The AVF evinced upregulation of p16Ink4a and p21Cip1 when assessed 3 days after AVF creation. The AVF also demonstrated increased SA-ß-gal activity in the artery and vein; staining for SA-ß-gal in the AVF artery, anastomosis, and vein; and a prominent SASP. Fisetin, an established senolytic that is protective in other models of vascular injury, when administered for 3 weeks, increased AVF blood flow and outward remodeling. Hemin, when administered for 3 weeks, decreased AVF blood flow. We demonstrate that hemin is a novel inducer of a senescence phenotype in endothelial cells, as reflected by several senescence indices. However, when administered relatively acutely (for 5 days) hemin increased AVF blood flow via HO-dependent mechanisms, as the latter was entirely prevented by a competitive inhibitor of HO activity. CONCLUSIONS: The rat AVF exhibits senescence within 3 days of its creation. Chronic administration of a senolytic compound (fisetin) increases AVF blood flow, whereas chronic administration of a pro-senescence compound (hemin) decreases AVF blood flow.

Increased production of superoxide anion contributes to dysfunction of the arteriovenous fistula.

Vascular access dysfunction causes morbidity in hemodialysis patients. This study examined the generation and pathobiological significance of superoxide anion in a rat femoral arteriovenous fistula (AVF). One week after AVF creation, there was increased production of superoxide anion accompanied by decreased total superoxide dismutase (SOD) and Cu/Zn SOD activities and induction of the redox-sensitive gene heme oxygenase-1. Immunohistochemical studies of nitrotyrosine formation demonstrated that peroxynitrite, a product of superoxide anion and nitric oxide, was present in increased amounts in endothelial and smooth muscle cells in the AVF. Because uncoupled NOS isoforms generate superoxide anion, and NOS coupling requires tetrahydrobiopterin (BH(4)) as a cofactor, we assessed NOS uncoupling by determining the ratio of BH(4) to dihydrobiopterin (BH(2)); the BH(4)-to-BH(2) ratio was markedly attenuated in the AVF. Because Src is a vasculopathic signaling species upstream and downstream of superoxide anion, such expression was evaluated; expression of Src and phosphorylated Src was both markedly increased in the AVF. Expression of NADPH oxidase (NOX) 1, NOX2, NOX4, cyclooxygenase (COX) 1, COX2, p47(phox), and p67(phox) was all unchanged, as assessed by Western analyses, thereby suggesting that these proteins may not be involved in increased production of superoxide anion. Finally, administration of tempol, a superoxide anion scavenger, decreased neointima formation in the juxta-anastomotic venous segment and improved AVF blood flow. We conclude that the AVF exhibits increased superoxide anion generation that may reflect the combined effects of decreased scavenging by SOD and increased generation by uncoupled NOS, and that enhanced superoxide anion production promotes juxta-anastomotic stenosis and impairs AVF function.

MCP-1 contributes to arteriovenous fistula failure.

Vascular access dysfunction compromises the care of patients on chronic hemodialysis. Elucidating the mechanisms of such dysfunction and devising strategies that may interrupt neointimal hyperplasia and relevant pathogenetic pathways are essential. Here, we show that, in the venous segment of a murine model of an arteriovenous fistula, monocyte chemoattractant protein-1 (MCP-1) mRNA and protein increase, accompanied by increased activity of the transcription factors NF-κB and AP-1. Genetic deficiency of MCP-1 proved markedly protective in this murine model, reflected by increased fistula patency 6 weeks after its formation, decreased venous wall thickness, and increased luminal area. An early effect of MCP-1 deficiency was the attenuation of the marked induction of CCL5 (RANTES) that occurred in this model, a chemokine recently recognized as a critical participant in vascular injury. Finally, in a rat model of an arteriovenous fistula, we localized expression of MCP-1 to the endothelium, proliferating smooth muscle cells and infiltrating leukocytes. In summary, marked upregulation of MCP-1 occurs in the venous segment of an arteriovenous fistula in rodents, and this vasculopathic chemokine contributes to failure of the fistula.

KLF11 Is a Novel Endogenous Protectant against Renal Ischemia-Reperfusion Injury.

Discovering new nephroprotectants may provide therapeutic strategies in AKI.This study provides the first evidence that KLF11, a member of the Krüppel-like factor (KLF) family of proteins, protects against AKI.In the absence of KLF11, exaggerated induction of endothelin-1 and IL-6 occurs after ischemic renal injury and may contribute to worse AKI.

The spike protein of SARS-CoV-2 induces heme oxygenase-1: Pathophysiologic implications.

BACKGROUND: Acute kidney injury (AKI) is both a consequence and determinant of outcomes in COVID-19. The kidney is one of the major organs infected by the causative virus, SARS-CoV-2. Viral entry into cells requires the viral spike protein, and both the virus and its spike protein appear in the urine of COVID-19 patients with AKI. We examined the effects of transfecting the viral spike protein of SARS-CoV-2 in kidney cell lines. METHODS: HEK293, HEK293-ACE2+ (stably overexpressing ACE2), and Vero E6 cells having endogenous ACE2 were transfected with SARS-CoV-2 spike or control plasmid. Assessment of gene and protein expression, and syncytia formation was performed, and the effects of quercetin on syncytia formation examined. FINDINGS: Spike transfection in HEK293-ACE2+ cells caused syncytia formation, cellular sloughing, and focal denudation of the cell monolayer; transfection in Vero E6 cells also caused syncytia formation. Spike expression upregulated potentially nephrotoxic genes (TNF-α, MCP-1, and ICAM1). Spike upregulated the cytoprotective gene HO-1 and relevant signaling pathways (p-Akt, p-STAT3, and p-p38). Quercetin, an HO-1 inducer, reduced syncytia formation and spike protein expression. INTERPRETATION: The major conclusions of the study are: 1) Spike protein expression in kidney cells provides a relevant model for the study of maladaptive and adaptive responses germane to AKI in COVID-19; 2) such spike protein expression upregulates HO-1; and 3) quercetin, an HO-1 inducer, may provide a clinically relevant/feasible protective strategy in AKI occurring in the setting of COVID-19. FUNDING: R01-DK119167 (KAN), R01-AI100911 (JPG), P30-DK079337; R01-DK059600 (AA).

Prominent Mitochondrial Injury as an Early Event in Heme Protein-Induced Acute Kidney Injury.

Background: Mitochondrial injury occurs in and underlies acute kidney injury (AKI) caused by ischemia-reperfusion and other forms of renal injury. However, to date, a comprehensive analysis of this issue has not been undertaken in heme protein-induced AKI (HP-AKI). We examined key aspects of mitochondrial function, expression of proteins relevant to mitochondrial quality control, and mitochondrial ultrastructure in HP-AKI, along with responses to heme in renal proximal tubule epithelial cells. Methods: The long-established murine glycerol model of HP-AKI was examined at 8 and 24 hours after HP-AKI. Indices of mitochondrial function (ATP and NAD+), expression of proteins relevant to mitochondrial dynamics, mitochondrial ultrastructure, and relevant gene/protein expression in heme-exposed renal proximal tubule epithelial cells in vitro were examined. Results: ATP and NAD+ content and the NAD+/NADH ratio were all reduced in HP-AKI. Expression of relevant proteins indicate that mitochondrial biogenesis (PGC-1α, NRF1, and TFAM) and fusion (MFN2) were impaired, as was expression of key proteins involved in the integrity of outer and inner mitochondrial membranes (VDAC, Tom20, and Tim23). Conversely, marked upregulation of proteins involved in mitochondrial fission (DRP1) occurred. Ultrastructural studies, including novel 3D imaging, indicate profound changes in mitochondrial structure, including mitochondrial fragmentation, mitochondrial swelling, and misshapen mitochondrial cristae; mitophagy was also observed. Exposure of renal proximal tubule epithelial cells to heme in vitro recapitulated suppression of PGC-1α (mitochondrial biogenesis) and upregulation of p-DRP1 (mitochondrial fission). Conclusions: Modern concepts pertaining to AKI apply to HP-AKI. This study validates the investigation of novel, clinically relevant therapies such as NAD+-boosting agents and mitoprotective agents in HP-AKI.

Characterization of a model of an arteriovenous fistula in the rat: the effect of L-NAME.

Vascular access dysfunction contributes to the mortality of patients undergoing chronic hemodialysis. The present study analyzed the changes that evolve in a femoral arteriovenous fistula in the rat. The venous segment of this model exhibited, at 1 week, activation of pro-inflammatory transcription factors and up-regulation of pro-inflammatory, proliferative, procoagulant, and profibrotic genes; and at 4 weeks, the venous segment displayed neointimal hyperplasia, smooth muscle proliferation, and thrombus formation. These changes were accompanied by endothelial (e) nitric oxide synthase (NOS) and inducible (i) NOS up-regulation. The administration of NG-nitro-L-arginine methyl ester, an inhibitor of NOS activity, increased venous neointimal hyperplasia and pro-inflammatory gene expression (monocyte chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant-1), increased systolic blood pressure, and decreased blood flow through the fistula. In another hypertensive model, the rat subtotal nephrectomy model, venous neointimal hyperplasia in the arteriovenous fistula was also exacerbated. We conclude that this arteriovenous fistula model recapitulates the salient features observed in dysfunctional, hemodialysis arteriovenous fistulas, and that venous neointimal hyperplasia is exacerbated when this model is superimposed in two different models of systemic hypertension. Since the uremic milieu contains increased amounts of asymmetric dimethylarginine, we speculate that such accumulation of this endogenous inhibitor of NOS, by virtue of its pressor or nitric oxide-depleting effects, or a combination thereof, may contribute to the limited longevity of arteriovenous fistulas used for hemodialysis.

Expression of ACE2 in the Intact and Acutely Injured Kidney.

Background: The actions of angiotensin-converting enzyme 2 (ACE2) oppose those of the renin-angiotensin-aldosterone system. ACE2 may be a cytoprotectant in some tissues. This study examined ACE2 expression in models of AKI. Methods: ACE2 mRNA and protein expression and ACE2 activity were assessed in murine ischemic AKI. Renal ACE2 mRNA expression was evaluated in LPS-induced AKI in wild-type (C57BL/6J) mice, in heme oxygenase-1+/+ and heme oxygenase-1-/- mice, and after unilateral ureteral obstruction (UUO) in wild-type mice. The effect of sex and age on renal ACE2 protein expression was also assessed. Results: In ischemic AKI, ACE2 mRNA and protein expression and ACE2 activity were reduced as compared with such indices in the intact kidney. In ischemic AKI, ACE2, which, in health, is prominently expressed in the tubular epithelium, especially proximal tubules, is decreased in expression in these segments. Decreased ACE2 expression in AKI did not reflect reduced GFR, because ACE2 mRNA expression was unaltered after UUO. LPS induced renal ACE2 mRNA expression in wild-type mice, but this effect did not occur in heme oxygenase-1-deficient mice. In ischemic and LPS-induced AKI, renal expression of the Mas receptor was increased. In the intact kidney, renal ACE2 protein expression decreased in female mice as compared with male mice, but was unaltered with age. Conclusion: We conclude that renal ACE2 expression is decreased in ischemic AKI, characterized by decreased GFR and abundant cell death, but is upregulated in LPS-induced AKI, an effect requiring heme oxygenase-1. Determining the significance of ACE2 expression in experimental AKI merits further study. We suggest that understanding the mechanism underlying ACE2 downregulation in AKI may offer insights relevant to COVID-19: ACE2 expression is downregulated after ACE2 mediates SARS-CoV-2 cellular entry; such downregulation is proinflammatory; and AKI commonly occurs and determines outcomes in COVID-19.

ß-Catenin is markedly induced in a murine model of an arteriovenous fistula: the effect of metalloproteinase inhibition.

Neointimal hyperplasia contributes to failure of hemodialysis arteriovenous fistulas (AVFs). Increased expression of matrix metalloproteinase (MMP)-9 occurs in AVFs, and MMP-9 is implicated in neointimal hyperplasia and vascular injury. Recent studies demonstrate that MMP-9, by degrading N-cadherin, leads to increased expression of ß-catenin and ß-catenin-dependent proliferation of smooth muscle cells. The present study examined this pathway in the venous limb of a murine AVF model. Western analyses demonstrate that, in this model, there is diminished expression of N-cadherin accompanied by increased expression of ß-catenin, c-Myc, and proliferating cell nuclear antigen (PCNA). By immunohistochemistry, ß-catenin and c-Myc localized to proliferating smooth muscle cells in the venous limb of the AVF. Increased expression of ß-catenin was accompanied by augmented expression of phosphorylated (p)-glycogen synthase kinase (GSK)-3ß, GSK-3ß, and integrin-linked kinase. The administration of doxycycline suppressed MMP-9 expression but did not reduce venous histological injury in the AVF, or increase AVF patency assessed 6 wk after its creation. Doxycycline did not influence expression of ß-catenin, c-Myc, GSK-3ß, or integrin-linked kinase. Thus, in this vascular injury model, the upregulation of ß-catenin cannot be readily attributed to MMP-9 upregulation; increased ß-catenin expression may reflect either the upregulation of p-GSK-3ß, GSK-3ß, or integrin-linked kinase. This study provides the first exploration of ß-catenin in an AVF, demonstrating substantial upregulation of this mitogenic signaling molecule and uncovering possible mechanisms that may account for such upregulation.

Induction of heme oxygenase-1 is a beneficial response in a murine model of venous thrombosis.

The induction of heme oxygenase-1 (HO-1) may protect against tissue injury. The present study examines the induction of HO-1 in a murine model of venous thrombosis and explores the downstream consequences of this induction. In a model of stasis-induced thrombosis created by ligation of the inferior vena cava, HO-1 expression is markedly induced. Such expression occurs primarily in smooth muscle cells in the venous wall and in leukocytes infiltrating the venous wall and clot. To determine the significance of HO-1 induction in venous thrombosis, this model was imposed in HO-1(+/+) and HO-1(-/-) mice. The initial clot size did not differ in either group by day 2, but was significantly larger in HO-1(-/-) mice by day 10, where an exaggerated inflammatory response in the venous wall was also observed. Following ligation of the inferior vena cava, HO-1(-/-) mice exhibited increased nuclear factor kappaB activation and markedly increased up-regulation of tissue factor, selectins, inflammatory cytokines, and matrix metalloproteinase-9, the latter incriminated in both clot lysis and vascular injury. We conclude that HO-1 deficiency impairs thrombus resolution and exaggerates the inflammatory response to thrombus formation. These findings offer insight into recent observations that polymorphisms in the HO-1 gene may increase the risk for recurrent venous thrombosis and dysfunction of hemodialysis arteriovenous fistulas, the latter caused, in part, by thrombosis.

L-4F, an apolipoprotein A-1 mimetic, restores nitric oxide and superoxide anion balance in low-density lipoprotein-treated endothelial cells.

BACKGROUND: Low-density lipoprotein (LDL) impairs endothelial cell function by uncoupling endothelial nitric oxide synthase (eNOS) activity, which allows superoxide anion (O2*-)) to be generated rather than nitric oxide (*NO). Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit the development of atherosclerotic lesions in LDL receptor-null mice. Here we hypothesize that L-4F, an apoA-1 mimetic that inhibits atherosclerosis induced by hypercholesterolemia, protects endothelial cell function by preventing LDL from uncoupling eNOS activity. METHODS AND RESULTS: Bovine aortic endothelial cells were incubated with LDL+/-L-4F, and changes in A23187-stimulated.NO and O2*- generation were determined by ozone chemiluminescence and superoxide dismutase-inhibitable ferricytochrome c reduction, respectively. Western analysis of eNOS immunoprecipitates was used to determine effects of LDL and L-4F on heat shock protein 90 (hsp90) interactions with eNOS. LDL decreased.NO production and increased eNOS-dependent O2*- generation. Pretreatment of LDL with L-4F increased.NO and decreased O2*- generation. By itself, L-4F had no effect on O2*- but did increase *NO generation. Stimulation of endothelial cells incubated with LDL decreased the association of hsp90 with eNOS. Pretreatment of LDL with L-4F prevented a decrease in hsp90 association with eNOS and often enhanced association on stimulation. CONCLUSIONS: These data demonstrate that L-4F protects endothelial cell function by preventing LDL from uncoupling eNOS activity. L-4F allows endothelial cell to maintain coupled eNOS activity to generate.NO even in the face of atherogenic concentrations of LDL.

Angiostatin: a negative regulator of endothelial-dependent vasodilation.

BACKGROUND: Angiostatin is known to inhibit certain aspects of endothelial function, eg, angiogenesis. Here we investigated the effects of angiostatin on another aspect of endothelial function, vasodilation, and examined mechanisms of inhibition--namely, association of heat-shock protein 90 (hsp90) with endothelial nitric oxide synthase (eNOS) and endothelial generation of nitric oxide (*NO) and superoxide anion (O2-). This avenue of investigation was based on recent reports suggesting that hsp90 modulates NOS production of *NO and O2-. METHODS AND RESULTS: Effects of angiostatin on vasodilation were determined in arterioles with the use of videomicroscopy in response to endothelium- and *NO-dependent vasodilators, acetylcholine (ACh) and vascular endothelial growth factor (VEGF), and an endothelium-independent agonist, papaverine. Association of hsp90 with eNOS was determined in rat aortas and bovine aortic endothelial cells (BAECs). Effects of angiostatin on *NO and O2- generation by BAECs were determined by ozone chemiluminescence and superoxide dismutase (SOD)--inhibitable ferricytochrome c reduction, respectively. Angiostatin impaired vasodilation mediated by ACh and VEGF but not papaverine. Pretreating arterioles with polyethylene glycolated--SOD (PEG-SOD) improved vasodilation to ACh and VEGF. Angiostatin decreased the association of hsp90 with eNOS in aortas and BAEC cultures and increased O2- generation in stimulated BAECs by an Lgamma-nitroargininemethylester (L-NAME)--inhibitable mechanism. CONCLUSIONS: These data indicate angiostatin alters endothelial function by allowing eNOS to generate O2- on activation. Such changes in enzyme function begin to explain, in part, why angiostatin is antiangiogenic and impairs endothelium-dependent vasodilation.

L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease.

BACKGROUND: Hypercholesterolemia and sickle cell disease (SCD) impair endothelium-dependent vasodilation by dissimilar mechanisms. Hypercholesterolemia impairs vasodilation by a low-density lipoprotein (LDL)-dependent mechanism. SCD has been characterized as a chronic state of inflammation in which xanthine oxidase (XO) from ischemic tissues increases vascular superoxide anion (O2*-) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor-null (Ldlr-/-) mice fed Western diets. Here we hypothesize that L-4F, an apoA-1 mimetic, preserves vasodilation in hypercholesterolemia and SCD by decreasing mechanisms that increase O2*- generation. METHODS AND RESULTS: Arterioles were isolated from hypercholesterolemic Ldlr-/- mice and from SCD mice that were treated with either saline or L-4F (1 mg/kg per day). Vasodilation in response to acetylcholine was determined by videomicroscopy. Effects of L-4F on LDL-induced increases in endothelium-dependent O2*- generation were determined on arterial segments via the hydroethidine assay and on stimulated endothelial cell cultures via superoxide dismutase-inhibitable ferricytochrome c reduction. Effects of L-4F on XO bound to pulmonary arterioles and content in livers of SCD mice were determined by immunofluorescence. Hypercholesterolemia impaired vasodilation in Ldlr-/- mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O2*- in arterial segments and in stimulated cultures. SCD impaired vasodilation, increased XO bound to pulmonary endothelium, and decreased liver XO content. L-4F dramatically improved vasodilation, decreased XO bound to pulmonary endothelium, and increased liver XO content compared with levels in untreated SCD mice. CONCLUSIONS: These data show that L-4F protects endothelium-dependent vasodilation in hypercholesterolemia and SCD. Our findings suggest that L-4F restores vascular endothelial function in diverse models of disease and may be applicable to treating a variety of vascular diseases.

Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity.

Dual increases in nitric oxide ((*)NO) and superoxide anion (O(2)(*-)) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of (*)NO generation to offset the increase in O(2)(*-) that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase (*)NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of (*)NO and O(2)(*-). Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 microM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated (*)NO production and increased L(omega)-nitroargininemethylester (L-NAME)-inhibitable O(2)(*-) generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O(2)(*-) generation, confirming that during proliferation eNOS generates (*)NO. Our findings demonstrate that hsp90 plays an important role in maintaining (*)NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit (*)NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.

Native low-density lipoprotein induces endothelial nitric oxide synthase dysfunction: role of heat shock protein 90 and caveolin-1.

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (*NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O(2)(*-)), which inactivates *NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for *NO, O(2)(*-), phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O(2)(*-) by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by approximately 34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (approximately 85%) and cytosol (approximately 15%). Only 3-6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O(2)(*-), which scavenges *NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O(2)(*-).