20‐Hydroxyeicosatetraenoic acid (20‐HETE) is a cytochrome p450‐derived eicosanoid that stimulates endothelial dysfunction and inflammation via binding to its receptor, GPR75 (GPCR‐Gαq/11). Increased 20‐HETE in animals and humans is associated with hypertension, stroke, myocardial infarction (MI), metabolic syndrome (MetS), and increased reactive oxygen species (ROS). However, investigated effectiveness of 20‐HETE antagonists on ROS generation and MI size has been limited by short‐term follow up and administration of inhibitors prior to onset of MI only in healthy animals. Here, we evaluated the effect of a 20‐HETE antagonist, 20‐SOLA, administered at onset of reperfusion on post MI remodeling 48 hours and 8 weeks after reperfusion in normal (SD) and MetS (JCR:LA‐cp) rats. 20‐HETE was elevated in response to ischemia and more so during reperfusion; this elevation was greater in MetS (ischemia: 2‐fold SD and JCR; reperfusion: 3‐fold SD, 4‐fold JCR). MI size was markedly greater in JCR vs. SD rate (50% vs. 25% of LV). Treatment with 20‐SOLA significantly decreased MI size in both SD (~50%) and JCR (~65%) rats. Equivalent results were obtained in animals treated with GPR75‐shRNA‐Lnv at onset of reperfusions. 20‐SOLA improved coronary blood flow (1.00 ± 0.01 to 1.84 ± 0.03 mg/ml/g in SD to 0.99 ± 0.03 to 1.75±0.01 mg/ml/g in JCR). Mechanistically, smaller MI size in 20‐SOLA‐treated animals correlated with decreased ROS production in JCR rats (90%). Furthermore, survival and left ventricular (LV) function were preserved 8 weeks post MI in 20‐SOLA‐treated animals (ejection fraction (EF) = 80.5% (SD, 100% survival) and 82.5% (JCR, 100% survival)). This correlated with decreased ROS, preserved myocyte morphology, and preserved intact collagen. Differential activation of MMPs in SD vs. JCR rats vs. 20‐SOLA‐treated animals underlie the observed morphological, structural and functional changes. ROS production was decreased in JCR rats treated with a NOX inhibitor (~60%); NOX inhibition also markedly reduced MI size at 48h (60%) and preserved LV function at 8 weeks (EF = 74%, 85% survival) vs. non‐treated rats demonstrating an important role for NOX‐derived ROS in determining MI size and long‐term post‐MI remodeling. In the aggregate, these results indicated that targeting 20‐HETE actions may be an important consideration in prevention of detrimental LV remodeling and mortality post MI. Funding: HL093052.Support or Funding InformationFunding: HL093052This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased intra‐abdominal (visceral) adipose tissue is a key feature of the metabolic syndrome. Expansion of visceral adipose tissue and the associated altered adipokine profile are linked to the development of hypertension. While reduction in visceral adipose tissue volume offers cardioprotective effects, the mechanisms underlying these effects remain understudied and unclear. In this study, we removed ~90% of visceral adipose tissue (=~5% body weight) by intra‐abdominal lipectomy and assessed large arterial stiffness, large artery structural matrix components, and blood pressure in a metabolic syndrome rat model (JCR:LA‐cp, JCR). Large artery compliance was significantly decreased in JCR vs. normal (Sprague Dawley, SD) rats (75±2% JCR vs. SD (carotid)) with a concomitant significant increase in MMP12‐dependent elastin degradation (3–6 fold vs. SD). Intra‐abdominal lipectomy normalized large artery stiffness, blocked MMP12 activation and reduced elastin degradation in JCR animals (~75% (carotid) vs. untreated JCR). Likewise, hypertension in JCR animals was significantly attenuated by intra‐abdominal lipectomy (MABP=156±3 mmHg JCR vs. 90±6 mmHg SD vs. 132±4 mmHg JCR+lipectomy). 20‐hydroxyeicosatetraeonic acid (20‐HETE), an arachidonic acid metabolite known to be a potent vasoconstrictor in resistance arteries, was significantly elevated in the visceral adipose tissue of JCR rats (~6 fold vs. SD). Like intra‐abdominal lipectomy, 20‐HETE antagonists restored large artery elasticity, blocked MMP12 activation and elastin degradation and significantly decreased blood pressure (125±3 mmHg JCR+20‐HETE antagonists) in JCR rats. Thus, 20‐HETE may be an important adipokine that mediates, in part, the adverse effects of expanded visceral fat volume in the metabolic syndrome. Its removal and inhibition may provide therapeutic and pharmacological approaches, respectively, for the management of central obesity‐driven large artery stiffness and hypertension in the metabolic syndrome.Support or Funding InformationNational Heart, Lung and Blood Institute (NHLBI): 1F31HL137356‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The authors regret to have made a mistake in the labelling of the y-axis in Fig. 1B and Figure VIB, Supplement. The y-axis should be in ng/mg protein. Also, the text of the corresponding figure legends as well as the text of Table 1, Supplement should read ng/mg protein instead of pg/mg protein. The authors would like to apologise for any inconvenience caused. Elevated 20-HETE in metabolic syndrome regulates arterial stiffness and systolic hypertension via MMP12 activationJournal of Molecular and Cellular CardiologyVol. 117PreviewArterial stiffness plays a causal role in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome P450 (CYP450)-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and loosely associated with obesity in humans. However, the role of 20-HETE in the regulation of large artery remodeling in metabolic syndrome has not been investigated. We hypothesized that elevated 20-HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. Full-Text PDF

Extracellular matrix (ECM) composition and regulation is an essential component of coronary collateral growth (CCG). The ECM serves several functions which include mechanical and structural support, cell‐to‐cell communication, and regulation of cell mobility and phenotype. Laminin is a major component of the ECM. In the metabolic syndrome, the ECM composition is altered. In this study, we investigated the role of laminin and its binding to the β6 integrin in the regulation of CCG in response to repetitive ischemia (RI), and cellular processes involved in CCG. Coronary blood flow in Sprague‐Dawley (SD) or metabolic syndrome rats (JCR:LA‐cp, JCR), was measured in the collateral (CZ) and normal zone (NZ) of the heart using microspheres. Collateral‐dependent blood flow in JCR rats was impaired compared to SD rats (CZ/NZ flow ratio was 0.11±0.03 in JCR vs. 0.85±0.04 in SD), which correlated with decreased expression of laminin (~5 fold) and of the β6 integrin (~6 fold) in JCR animals. Administration of blocking‐antibodies against laminin or the β6 integrin in SD rats completely blocked CCG and decreased each other's expression, respectively. Furthermore, both laminin and β6 integrin expression were increased in endothelial cells (ECs) subjected to cycles of hypoxia‐hyperoxia‐normoxia to mimic RI vs. ECs cultured in normoxia. Blocking antibodies against laminin or the β6 integrin significantly impaired EC proliferation, migration and tube formation (~2–2.5 fold). TGF‐β signaling through Smad2/3 and leading to MMP 2/9 production, which is required for successful CCG, was also impaired (90%). Accordingly, TGF‐β receptor inhibition completely blocked CCG. Taken together, these results point to an important TGF‐β‐mediated pathway which promotes CCG and the activation of which is dependent on expression and the interaction between laminin and the β6 integrin. Support or Funding Information Support: NIH R01 HL093052 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Arterial stiffness plays a causal role in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome P450 (CYP450)-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and loosely associated with obesity in humans. However, the role of 20-HETE in the regulation of large artery remodeling in metabolic syndrome has not been investigated. We hypothesized that elevated 20-HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. 20-HETE production was increased ~7 fold in large, conduit arteries of metabolic syndrome (JCR:LA-cp, JCR) vs. normal Sprague-Dawley (SD) rats. This correlated with increased elastin degradation (~7 fold) and decreased arterial compliance (~75% JCR vs. SD). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. 20-HETE antagonists normalized, and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly improved (~50% vs. untreated JCR) large artery compliance in JCR rats. 20-HETE antagonists also decreased systolic (182 ± 3 mmHg JCR, 145 ± 3 mmHg JCR + 20-HETE antagonists) but not diastolic blood pressure in JCR rats. Whereas diastolic pressure was fully angiotensin II (Ang II)-dependent, systolic pressure was only partially Ang II-dependent, and large artery stiffness was Ang II-independent. Thus, 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high 20-HETE production in large, conduit arteries, which results in increased large artery stiffness and systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.

Large artery stiffness is a causal factor in development of systolic hypertension. 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome CYP450-derived arachidonic acid metabolite, is known to be elevated in resistance arteries in hypertensive animal models and in obesity in humans, but the role of 20-HETE in regulation of large artery remodeling in metabolic syndrome has not been investigated. Unlike normal (Sprague-Dawley (SD)) rats, large arteries (aorta, carotid and >100μM mesenteric arteries) of metabolic syndrome rats (JCR:LA-cp, JCR) express CYP4A and 4F, CYP450s which make 20-HETE in rats (2-fold increase vs. SD). Consequently, 20-HETE production is elevated in large arteries of JCR rats. We hypothesized that this elevated 20-HETE increases matrix metalloproteinase 12 (MMP12, an elastase) activation leading to increased degradation of elastin, increased large artery stiffness and increased systolic blood pressure. A 3-4 fold increase in 20-HETE production in large arteries of JCR vs. SD rats correlated with increased elastin degradation (3-6 fold) and increased arterial stiffness (~75%). 20-HETE antagonists blocked elastin degradation in JCR rats concomitant with blocking MMP12 activation. Importantly, 20-HETE antagonists and MMP12 inhibition (pharmacological and MMP12-shRNA-Lnv) significantly decreased (~60% vs. untreated JCR) large artery stiffness in JCR rats. 20-HETE antagonists also decreased systolic (182±3 mmHg JCR, 145±3 mmHg JCR+20-HETE antagonists) but not diastolic (125±4 mmHg JCR, 124±4 mmHg JCR+20-HETE antagonists) blood pressure in JCR rats. Whereas diastolic pressure was fully angiotensin II (Ang II)-dependent, systolic pressure was only partially Ang II-dependent, and large artery stiffness in JCR rats was Ang II-independent. These results suggest that 20-HETE-dependent regulation of systolic blood pressure may be a unique feature of metabolic syndrome related to high CYP4A/4F expression and resultant high 20-HETE production in large conduit arterial stiffness, which is a primary determinant of systolic blood pressure. These findings may have implications for management of systolic hypertension in patients with metabolic syndrome.

Thirty percent of the world population is diagnosed with metabolic syndrome. High-fat/high-sucrose (HF/HS) diet (Western diet) correlates with metabolic syndrome prevalence. We characterized effects of the HF/HS diet on vascular (arterial stiffness, vasoreactivity, and coronary collateral development) and cardiac (echocardiography) function, oxidative stress, and inflammation in a rat model of metabolic syndrome (JCR rats). Furthermore, we determined whether male versus female animals were affected differentially by the Western diet. Cardiovascular function in JCR male rats was impaired versus normal Sprague-Dawley (SD) rats. HF/HS diet compromised cardiovascular (dys) function in JCR but not SD male rats. In contrast, cardiovascular function was minimally impaired in JCR female rats on normal chow. However, cardiovascular function in JCR female rats on the HF/HS diet deteriorated to levels comparable to JCR male rats on the HF/HS diet. Similarly, oxidative stress was markedly increased in male but not female JCR rats on normal chow but was equally exacerbated by the HF/HS diet in male and female JCR rats. These results indicate that the Western diet enhances oxidative stress and cardiovascular dysfunction in metabolic syndrome and eliminates the protective effect of female sex on cardiovascular function, implying that both males and females with metabolic syndrome are at equal risk for cardiovascular disease.NEW & NOTEWORTHY Western diet abolished protective effect of sex against cardiovascular disease (CVD) development in premenopausal animals with metabolic syndrome. Western diet accelerates progression of CVD in male and female animals with preexisting metabolic syndrome but not normal animals. Exacerbation of baseline oxidative stress correlates with accelerated progression of CVD in metabolic syndrome animals on Western diet.

Increased intra-abdominal (visceral) adipose tissue is a key feature of the metabolic syndrome affecting over 30% of the U.S. population. Expansion of visceral adipose tissue is linked to the development of hypertension and is a risk factor for cardiovascular disease that can ultimately lead to end-organ damage. While reduction in visceral adipose tissue volume offers cardioprotective effects, the cardiovascular mechanisms behind these beneficial effects remain unclear. In this study, we removed ~90% of visceral adipose tissue (=~5% body weight) by intra-abdominal lipectomy and assessed large arterial stiffness, large artery structural matrix components, and blood pressure in a metabolic syndrome rat model (JCR:LA-cp, JCR). Large artery stiffness was significantly elevated in JCR vs. normal (Sprague Dawley, SD) rats (75±2% JCR vs. SD (carotid)) with a concomitant significant increase in MMP12-dependent elastin degradation (3-6 fold vs. SD). Intra-abdominal lipectomy normalized large artery stiffness, blocked MMP12 activation and reduced elastin degradation in JCR animals (~75% (carotid) vs. untreated JCR). Likewise, hypertension in JCR animals was significantly attenuated by intra-abdominal lipectomy (MABP=156±3 mmHg JCR vs. 90±6 mmHg SD vs. 132±4 mmHg JCR+lipectomy). 20-hydroxyeicosatetraeonic acid (20-HETE), an arachidonic acid metabolite known to be a potent vasoconstrictor in resistance arteries, was significantly elevated in the visceral adipose tissue of JCR rats (~6 fold vs. SD). Intra-abdominal lipectomy normalized 20-HETE levels in JCR rats. Like intra-abdominal lipectomy, 20-HETE antagonists restored large artery elasticity, blocked MMP12 activation and elastin degradation, and significantly decreased blood pressure (125±3 mmHg JCR+20-HETE antagonists) in JCR rats. Thus, 20-HETE may be an important adipokine that mediates the adverse effects of expanded visceral fat volume in the metabolic syndrome and its inhibition may provide a pharmacological approach for the management of central obesity-driven large artery stiffness and hypertension.

Coronary collateral growth (CCG) is impaired in metabolic syndrome (MetS). microRNA-145 (miR-145-Adv) delivery to our rat model of MetS (JCR) completely restored and neutrophil depletion significantly improved CCG. We determined whether low endogenous levels of miR-145 in MetS allowed for elevated production of 20-hydroxyeicosatetraenoic acid (20-HETE), which, in turn, resulted in excessive neutrophil accumulation and endothelial dysfunction leading to impaired CCG. Rats underwent 0-9 days of repetitive ischemia (RI). RI-induced cardiac CYP4F (neutrophil-specific 20-HETE synthase) expression and 20-HETE levels were increased (4-fold) in JCR vs. normal rats. miR-145-Adv and 20-HETE antagonists abolished and neutrophil depletion (blocking antibodies) reduced (~60%) RI-induced increases in CYP4F expression and 20-HETE production in JCR rats. Impaired CCG in JCR rats (collateral-dependent blood flow using microspheres) was completely restored by 20-HETE antagonists [collateral-dependent zone (CZ)/normal zone (NZ) flow ratio was 0.76 ± 0.07 in JCR + 20-SOLA, 0.84 ± 0.05 in JCR + 20-HEDGE vs. 0.11 ± 0.02 in JCR vs. 0.84 ± 0.03 in normal rats]. In JCR rats, elevated 20-HETE was associated with excessive expression of endothelial adhesion molecules and neutrophil infiltration, which were reversed by miR-145-Adv. Endothelium-dependent vasodilation of coronary arteries, endothelial nitric oxide synthase (eNOS) Ser1179 phosphorylation, eNOS-dependent NO·- production and endothelial cell survival were compromised in JCR rats. These parameters of endothelial dysfunction were completely reversed by 20-HETE antagonism or miR-145-Adv delivery, whereas neutrophil depletion resulted in partial reversal (~70%). We conclude that low miR-145 in MetS allows for increased 20-HETE, mainly from neutrophils, which compromises endothelial cell survival and function leading to impaired CCG. 20-HETE antagonists could provide viable therapy for restoration of CCG in MetS.NEW & NOTEWORTHY Elevated 20-hydroxyeicosatetraenoic acid (20-HETE) impairs coronary collateral growth (CCG) in metabolic syndrome by eliciting endothelial dysfunction and apoptosis via excessive neutrophil infiltration. 20-HETE antagonists completely restore coronary collateral growth in metabolic syndrome. microRNA-145 (miR-145) is an upstream regulator of 20-HETE production in metabolic syndrome; low expression of miR-145 in metabolic syndrome promotes elevated production of 20-HETE.

Central (visceral) obesity is a key feature of the metabolic syndrome and an independent predictor of cardiovascular disease. Reux en Y gastric bypass (RnY) has been shown to offer protection against cardiovascular disease, but residual risk remains. It is also unknown whether the cardiovascular benefit is a consequence of a decrease in visceral (intra-abdominal) adipose tissue or of other factors. In this study, we compared the effects of RnY vs. removal of 90% of visceral adipose tissue (=5% body weight) by intra-abdominal lipectomy on cardiac function (echocardiography), macrovascular function (carotid artery stiffness) and microvascular function (coronary artery endothelium-dependent vasorelaxation) in a metabolic syndrome rat model (JCR:LA-cp, JCR). Cardiac output (CO) and ejection fraction (EF) were significantly decreased in JCR vs. normal (Sprague-Dawley, SD) rats (CO=50±5%, EF=45±2% of normal), and were significantly improved by both RnY and intra-abdominal lipectomy (CO=75±6%, EF=82±2% and CO=80±3%, EF=90±2% of normal, respectively). Likewise, acetylcholine-dependent coronary artery vasorelaxation was impaired in JCR rats (50±1% of normal), and was significantly improved by both RnY and intra-abdominal lipectomy (98±2% and 98±3% of normal, respectively). Carotid artery stiffness was significantly increased in JCR rats (~2 fold vs. SD), and was normalized by intra-abdominal lipectomy (to equal SD), but not by RnY (~2 fold vs. SD). Intra-abdominal lipectomy but not RnY also decreased cardiac and vascular elastin degradation in JCR rats (Lipectomy: ~50% (heart), ~75% (carotid); RnY: ~15% (heart), ~5% (carotid) vs. untreated JCR, respectively), concomitant with a decrease in matrix metalloproteinase 12 (MMP12), a major elastase, activation (~50% (heart), ~75% (carotid), ~87% (visceral fat), ~75% (circulating) vs. untreated JCR) and in 20-hydroxyeicosatetraeonic acid (20-HETE) levels (~4 (heart), ~7 (carotid), ~4 (visceral fat), ~4 (circulating) fold vs. untreated JCR). Thus, our data indicate that intra-abdominal adipose tissue itself is a source of factors that may be important negative regulators of micro- and macrovascular and cardiac function, but are not eliminated by RnY.

Transient, repetitive myocardial ischemia (RI)-induced coronary collateral growth (CCG) is impaired in metabolic syndrome patients and animal models. Endothelial cell (EC) dysfunction and chronic inflammation are hallmarks of metabolic syndrome. We showed that while in normal animals (SD), RI induces transient infiltration of monocytes, associated with successful CCG, in metabolic syndrome rats (JCR), RI induces sustained accumulation of neutrophils, which contributes to compromised CCG. 20-hydroxyeicosatetraeonic acid (20-HETE) is a pro-inflammatory metabolite of arachidonic acid. Its role in the regulation of CCG is unknown. We hypothesized that enhanced 20-HETE-mediated neutrophil adhesion to ECs and consequent EC dysfunction and apoptosis result in impaired CCG in metabolic syndrome. P-selectin and ICAM-1 expression was increased ~40% in JCR vs. SD rats. This increase was prevented by 20-HETE antagonists, 20-SOLA or 20-HEDGE. 20-HETE antagonists also prevented neutrophil accumulation observed in JCR rats. Coronary arteries from JCR rats exhibited reduced endothelium (Ach)-dependent vasodilation (20% JCR vs. 50% of max. SD). RI-induced eNOS activation and NO production were likewise decreased (~60% and~70%, respectively) in JCR vs. SD rats. EC apoptosis (TUNEL) was severely increased in response to RI in JCR rats (~75% vs. SD). Neutrophil adhesion-blocking antibodies partially attenuated EC apoptosis (~70%) and EC dysfunction (~75% eNOS activation and NO production, 75% Ach-dependent vasodilation). 20-HETE antagonists fully reversed impaired endothelium-dependent vasodilation, eNOS activation, NO production and prevented EC apoptosis. Finally, impaired CCG in JCR rats (collateral-dependent blood flow, microspheres) was completely restored by 20-HETE antagonists (CZ/NZ flow was 0.76±0.07 in JCR+20-SOLA, 0.84±0.05 in JCR+20-HEDGE vs. 0.11±0.02 in JCR vs. 0.84±0.03 ml/min/g in SD rats) and partially restored by neutrophil-blocking antibodies (0.49±0.05 ml/min/g). Taken together, these results indicate that 20-HETE-dependent neutrophil adhesion and accumulation compromises EC survival and function leading to impaired CCG. 20-HETE antagonists could provide therapy for restoration of CCG in metabolic syndrome.

Coronary collateral growth (CCG) is an adaptive response to transient, repetitive coronary artery occlusion in normal animals but is markedly impaired in metabolic syndrome. Recent studies in our laboratory have shown that in metabolic syndrome rats (JCR:LA‐cp, JCR) in addition to endothelial cell (EC) dysfunction, neutrophil survival is enhanced, leading to neutrophil accumulation which contributes to compromised CCG in JCR rats. 20‐hydroxyeicosatetraenoic acid (20‐HETE) plays an important role in vascular function. However, the role of 20‐HETE in the regulation of CCG is unknown. We investigated the hypothesis that elevated neutrophil‐derived 20‐HETE promotes EC dysfunction and apoptosis and impairs CCG in metabolic syndrome. In our study, coronary blood flow in Sprague‐Dawley (SD) or JCR rats was measured in the collateral (CZ) and normal zone (NZ) of the heart after 9 days of transient, repetitive LAD occlusion (repetitive ischemia, RI) using microspheres. Collateral growth in the JCR rats was impaired compared to SD rats (CZ/NZ flow ratio was 0.11±0.02 in JCR vs. 0.84±0.02 in SD). Administration of 20‐HETE antagonists, either 20‐SOLA or 20‐HEDGE, fully restored collateral‐dependent blood flow (0.85±0.01 in JCR+20‐SOLA, 0.84±0.01 in JCR+20‐HEDGE), while neutrophil depletion, via neutrophil‐blocking antibodies, partially restored collateral‐dependent blood flow (0.57±0.02) in JCR rats. Neutrophil accumulation was ~3 times higher and persisted for the duration of the RI protocol in the CZ of JCR vs. SD rats, where their infiltration was low and transient. This RI‐induced, elevated neutrophil infiltration in the CZ of JCR rats correlated with a >4‐fold increase in CYP4F (the neutrophil 20‐HETE CYP isoform) expression and a 4‐fold increase in 20‐HETE levels vs. SD rats, which was abolished by administration of neutrophil‐blocking antibodies to JCR rats. Furthermore, coronary microvessels of JCR rats displayed reduced endothelium‐dependent vasodilation after 9 days of RI (20%±1.2% JCR vs. 40%±2.3% SD), which was reversed with neutrophil‐blocking antibodies (37%±2.5% JCR+anti‐CD11b/CD18/CD44). eNOS phosphorylation (p1179) and NO production were likewise decreased (~50%) in the CZ of JCR rays after 9 days of RI, which was reversed after administration of neutrophil‐blocking antibodies. Lastly, RI‐induced EC apoptosis in JCR but not in SD rats (TUNEL). EC viability in JCR rats was almost completely restored by treatment with neutrophil‐blocking antibodies. Taken together, these results indicate that neutrophil‐derived 20‐HETE is a major contributor to impaired EC viability and function, and thus to impaired CCG in metabolic syndrome.Support or Funding InformationSupport: NIH R01 HL093052.

Hypertension is a risk factor that is associated with the metabolic syndrome. Large artery stiffness, which is in part a result of elastin degradation, plays a causal role in the development of isolated systolic hypertension, which is especially common in the elderly. Levels of 20‐hydroxyeicosatetraeonic acid (20‐HETE), a cytochrome (CYP)‐derived arachidonic acid metabolite, are greatly elevated in hypertensive animal models and loosely associated with obesity in humans, but 20‐HETE's role in the regulation of arterial stiffness in the metabolic syndrome has not been investigated. We hypothesized that elevated 20‐HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased elastin degradation, increased large artery stiffness and increased systolic blood pressure. Our study shows that elastin degradation was increased ~4 fold in large arteries of metabolic syndrome rats (JCR:LA‐cp, JCR) vs. Sprague‐Dawley (SD) control rats. This correlated with increased large artery stiffness (75%±2% JCR vs. SD). 20‐SOLA (2,5,8,11,14,17‐hexaoxanonadecan‐19‐yl 20‐hydroxyicosa‐6( Z ),15( Z )‐dienoate) a 20‐HETE antagonist, blocked elastin degradation in JCR rats, concomitant with decreasing MMP12 activation. Importantly, both 20‐SOLA and MMP12 inhibition (pharmacological and MMP12‐shRNA‐Lnv) decreased large artery stiffness in JCR rats (3%±2% JCR+20‐SOLA vs. SD, 18%±4.3% JCR+MMP12 inhibition vs. SD). 20‐SOLA and MMP12 inhibition also decreased systolic but not diastolic blood pressure in JCR rats (113±6 mmHg SD, 159±4 mmHg JCR, 130±4 mmHg JCR+20‐SOLA, 139±5 JCR+MMP12 mmHg inhibition). These data suggest that a portion of the systolic but not diastolic component of hypertension in the metabolic syndrome JCR rats is 20‐HETE‐sensitive and dependent on large artery stiffness/compliance. The source(s) of 20‐HETE and MMP12 responsible for increased elastin degradation and large artery stiffness are likewise unknown. Intra‐abdominal lipectomy (removal of visceral fat=5% body weight) in JCR rats decreased MMP12 activation, elastin degradation and large artery stiffness to levels achieved with 20‐SOLA and observed in SD control rats, suggesting: 1) that this 20‐HETE is largely derived from visceral (intra‐abdominal) adipose tissue, and 2) that MMP12 involved in the regulation of vascular stiffness is also derived primarily from visceral adipose tissue. Future implications of these findings may be important for systolic hypertension management through 20‐HETE and/or MMP12 inhibition. Support or Funding Information Supported by NIH R01HL093052.

Transient, repetitive myocardial ischemia (RI)-induced coronary collateral growth (CCG) is impaired in metabolic syndrome patients and animal models. Endothelial cell (EC) dysfunction and chronic inflammation are hallmarks of metabolic syndrome. We showed that while in normal animals (SD), RI induces transient infiltration of monocytes, associated with successful CCG, in metabolic syndrome rats (JCR), RI induces sustained accumulation of neutrophils, which contributes to compromised CCG. 20-hydroxyeicosatetraeonic acid (20-HETE) is a pro-inflammatory metabolite of arachidonic acid. Its role in the regulation of CCG is unknown. We hypothesized that enhanced 20-HETE-mediated neutrophil adhesion to ECs and consequent EC dysfunction and apoptosis result in impaired CCG in metabolic syndrome. P-selectin and ICAM-1 expression was increased ~40% in JCR vs. SD rats. This increase was prevented by 20-HETE antagonists, 20-SOLA or 20-HEDGE. 20-HETE antagonists also prevented neutrophil accumulation observed in JCR rats. Coronary arteries from JCR rats exhibited reduced endothelium (Ach)-dependent vasodilation (20% JCR vs. 50% of max. SD). RI-induced eNOS activation and NO production were likewise decreased (~60% and~70%, respectively) in JCR vs. SD rats. EC apoptosis (TUNEL) was severely increased in response to RI in JCR rats (~75% vs. SD). Neutrophil adhesion-blocking antibodies partially attenuated EC apoptosis (~70%) and EC dysfunction (~75% eNOS activation and NO production, 75% Ach-dependent vasodilation). 20-HETE antagonists fully reversed impaired endothelium-dependent vasodilation, eNOS activation, NO production and prevented EC apoptosis. Finally, impaired CCG in JCR rats (collateral-dependent blood flow, microspheres) was completely restored by 20-HETE antagonists (CZ/NZ flow was 0.76±0.07 in JCR+20-SOLA, 0.84±0.05 in JCR+20-HEDGE vs. 0.11±0.02 in JCR vs. 0.84±0.03 ml/min/g in SD rats) and partially restored by neutrophil-blocking antibodies (0.49±0.05 ml/min/g). Taken together, these results indicate that 20-HETE-dependent neutrophil adhesion and accumulation compromises EC survival and function leading to impaired CCG. 20-HETE antagonists could provide therapy for restoration of CCG in metabolic syndrome.

Coronary collateral growth (CCG) is impaired in metabolic syndrome. microRNA-21 (miR-21) is a proproliferative and antiapoptotic miR, which we showed to be elevated in metabolic syndrome. Here we investigate whether impaired CCG in metabolic syndrome involved miR-21-mediated aberrant apoptosis. Normal Sprague-Dawley (SD) and metabolic syndrome [J. C. Russel (JCR)] rats underwent transient, repetitive coronary artery occlusion [repetitive ischemia (RI)]. Antiapoptotic Bcl-2, phospho-Bad, and Bcl-2/Bax dimers were increased on days 6 and 9 RI, and proapoptotic Bax and Bax/Bax dimers and cytochrome-c release concurrently decreased in JCR versus SD rats. Active caspases were decreased in JCR versus SD rats (~50%). Neutrophils increased transiently on day 3 RI in the collateral-dependent zone of SD rats but remained elevated in JCR rats, paralleling miR-21 expression. miR-21 downregulation by anti-miR-21 induced neutrophil apoptosis and decreased Bcl-2 and Bcl-2/Bax dimers (~75%) while increasing Bax/Bax dimers, cytochrome-c release, and caspase activation (~70, 400, and 400%). Anti-miR-21 also improved CCG in JCR rats (~60%). Preventing neutrophil infiltration with blocking antibodies resulted in equivalent CCG recovery, confirming a major role for deregulated neutrophil apoptosis in CCG impairment. Neutrophil and miR-21-dependent CCG inhibition was in significant part mediated by increased oxidative stress. We conclude that neutrophil apoptosis is integral to normal CCG and that inappropriate prolonged miR-21-mediated survival of neutrophils plays a major role in impaired CCG, in part via oxidative stress generation.

We have previously shown that transient and repetitive ischemia-induced (RI) coronary collateral growth (CCG) was severely impaired in a metabolic syndrome rat model (JCR rat). Levels of 20-hydroxyeicosatetraeonic acid (20-HETE), a cytochrome (CYP)-derived arachidonic acid metabolite are greatly elevated in hypertensive animal models and loosely associated with obesity in humans, but its levels in metabolic syndrome, especially in cardiovascular tissues, as well as its possible involvement in the regulation of collateral growth are unknown. In rats, CYP4A1 is the major enzyme responsible for the production of 20-HETE. In this study, we demonstrated that cardiac CYP4A1 expression (RT-PCR, Western blot and immunohistochemistry) and 20-HETE levels were markedly (10-fold) elevated in JCR vs. Sprague-Dawly (SD) rats in response to RI. Importantly, administration of an antagonist of 20-HETE, 20-SOLA, completely restored CCG in JCR rats (collateral flow was 86±1% of that in the normal zone (JCR+SOLA) vs. 21±2% (JCR) vs. 84±5% (SD), p<0.05). We conclude that 20-HETE is an important modulator of CCG in the metabolic syndrome where its myocardial tissue levels are highly elevated.

Inadequate cell proliferation is considered a major causative factor for impaired coronary collateral growth (CCG). Proangiogenic growth factors (GFs) stimulate cell proliferation, but their administration does not promote CCG in patients. These GFs are increased in patients with metabolic syndrome and in animal models, where CCG is impaired. Here, we investigated whether excessive cell proliferation underlies impaired CCG in metabolic syndrome. Normal [Sprague‐Dawley (SD)] and metabolic syndrome [James C. Russell (JCR)] rats underwent repetitive ischemia (RI; transient, repetitive coronary artery occlusion and myocardial ischemia). We have shown that CCG was maximal at d 9 of RI in SD rats but did not occur in JCR rats. The increase in cell proliferation (PCNA, Ki‐67, cyclin A, phospho‐ cdc2, p21Waf, p27Kip) was transient (~4‐fold, d 3 RI) in SD rats but greater and sustained in JCR rats (~8‐ to 6‐fold, d 3‐9 RI). In JCR rats, this was associated with increased and sustained miR‐21 expression and accumulation of proliferating synthetic vascular smooth muscle cells in the lumen of small arterioles, which failed to undergo outward expansion. Administration of anti‐miR‐21 blocked RI‐induced cell proliferation and significantly improved CCG in JCR rats (~60%). miR‐21‐dependent excessive cell proliferation in the later stages of collateral remodeling correlates with impaired CCG in metabolic syndrome.—Hutcheson, R., Chaplin, J., Hutcheson, B., Borthwick, F., Proctor, S., Gebb, S., Jadhav, R., Smith, E., Russell, J. C., Rocic, P. miR‐21 normalizes vascular smooth muscle proliferation and improves coronary collateral growth in metabolic syndrome. FASEB J. 28, 4088‐4099 (2014). www.fasebj.org

Rebecca Hutcheson,* Russell Terry,* Brenda Hutcheson, Rashmi Jadhav, Jennifer Chaplin, Erika Smith, Robert Barrington, Spencer D. Proctor, and Petra Rocic Department of Pharmacology, New York Medical College, Valhalla, New York; Department of Biochemistry and Molecular Biology, University of South Alabama College of Medicine, Mobile, Alabama; Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama; and Metabolic and Cardiovascular Diseases Laboratory, Alberta Institute for Human Nutrition, University of Alberta, Edmonton, Alberta, Canada