A Hydrogel Strategy to Augment Tissue Adenosine to Improve Hindlimb Perfusion.

[Figure: see text].

5,10-methylenetetrahydrofolate reductase C677T gene polymorphism and peripheral arterial disease: A meta-analysis.

INTRODUCTION: Peripheral arterial disease is one common vascular disease most caused by atherosclerosis. As with stroke and coronary heart disease, peripheral arterial disease is one clinical type of atherosclerotic cardiovascular disease with many unmeasured environmental and genetic components. MTHFR C677T polymorphism is associated with the increased risk of ischemic stroke and coronary heart disease. MTHFR C677T polymorphism is associated with decreasing enzyme activity and increasing homocysteine levels. Meta-analysis of studies had demonstrated an association between elevated plasma homocysteine levels and peripheral arterial disease. Elevated plasma homocysteine level is closely related to MTHFR C677T polymorphism. Recent studies had clarified the relationship of MTHFR C677T polymorphism and peripheral arterial disease. So we performed a meta-analysis to investigate the association between MTHFR C677T polymorphism and peripheral arterial disease. MATERIALS AND METHODS: We searched the database PubMed, Embase, and Cochrane Library for all English-language articles related to peripheral arterial disease and MTHFR C677T through 30 June 2020. Analysis results were shown by forest plot. Publication bias was estimated using funnel plot. RESULTS: A total of 15 studies comprising 1929 patients with peripheral arterial disease and 2952 healthy controls were included in the meta-analysis. Significant associations between MTHFR C677T genetic polymorphism and peripheral arterial disease were found (OR = 1.31, 95% CI: 1.09-1.58, P <0.01). But there was no significant association (poor OR = 1.11, 95% CI: 0.98-1.26, P =0.11) between the T allele carrier and peripheral arterial disease. CONCLUSION: Our meta-analysis suggested that MTHFR C677T genetic polymorphism TT genotype may be associated with increased peripheral arterial disease risk. But further studies with large sample sizes are needed to confirm our findings.

Skeletal muscle expression of adipose-specific phospholipase in peripheral artery disease.

Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.

Dysregulation of FOXO1 (Forkhead Box O1 Protein) Drives Calcification in Arterial Calcification due to Deficiency of CD73 and Is Present in Peripheral Artery Disease.

OBJECTIVE: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. CONCLUSIONS: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.

Lipoprotein-associated phospholipase A2 predicts lower limb ischemia in hemodialysis subjects.

Hemodialyzed patients (HD) have high prevalence of peripheral arterial disease. In the general population, lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with peripheral arterial disease but no data are available for renal subjects. The aim of this study was to evaluate the relationship between Lp-PLA2 and lower limb ischemia among dialyzed patients. One hundred and two dialyzed subjects, with median (IQR) age of 71 (59-78) years, enrolled in June 2013 and followed until June 2018, were investigated for Lp-PLA2 activity and the occurrence of peripheral arterial disease and lower limb ischemia. The median (IQR) levels of Lp-PLA2 were 184 nmol/min/mL (156.5-214.5). The 43 HD patients with abnormal Lp-PLA2 activity (>194 nmol/min/mL) had higher levels of total and LDL-cholesterol, ApoB/A1 ratio, and higher occurrence of lower limb ischemia during the follow up (44% vs 17%, P = .003). In multivariate analysis, Lp-PLA2 activity (P = .018) and diabetes (P < .001) were independently associated with time to lower limb ischemia, and when the presence of previous PAD was added to the multivariate model, only presence of previous PAD (P < .001) and Lp-PLA2 (P = .003) remained associated. Lp-PLA2 is an independent predictor of lower limb ischemia in dialyzed patients.

Ablation of angiotensin type 2 receptor prevents endothelial nitric oxide synthase glutathionylation and nitration in ischaemic abductor muscle of diabetic mice.

Peripheral artery disease is a severe complication of diabetes. We have reported that the deletion of angiotensin type 2 receptor in diabetic mice promoted vascular angiogenesis in the ischaemic muscle 4 weeks following ischaemia. However, the angiotensin type 2 receptor deletion beneficial effects occurred 2 weeks post surgery suggesting that angiotensin type 2 receptor may regulate other pro-angiogenic signalling pathways during the early phases of ischaemia. Nondiabetic and diabetic angiotensin type 2 receptor-deficient mice (Agtr2-/Y) underwent femoral artery ligation after 2 months of diabetes. Blood perfusion was measured every week up to 2 weeks post surgery. Expression of vascular endothelial growth factor, vascular endothelial growth factor receptor and endothelial nitric oxide synthase expression and activity were evaluated. Blood flow reperfusion in the ischaemic muscle of diabetic Agtr2+/Y mice was recovered at 35% as compared to a 68% recovery in diabetic Agtr2-/Y mice. The expression of vascular endothelial growth factor and its receptors was diminished in diabetic Agtr2+/Y mice, an observation not seen in diabetic Agtr2-/Y mice. Interestingly, Agtr2-/Y mice were protected from diabetes-induced glutathionylation, nitration and decreased endothelial nitric oxide synthase expression, which correlated with reduced endothelial cell death and enhanced vascular density in diabetic ischaemic muscle. In conclusion, our results suggest that the deletion of angiotensin type 2 receptor promotes blood flow reperfusion in diabetes by favouring endothelial cell survival and function.

Dual specificity phosphatase 5 regulates perfusion recovery in experimental peripheral artery disease.

Peripheral artery disease (PAD) is caused by atherosclerotic occlusions of vessels outside the heart, particularly those of the lower extremities. Angiogenesis is one critical physiological response to vessel occlusion in PAD, but our understanding of the molecular mechanisms involved in angiogenesis is incomplete. Dual specificity phosphatase 5 (DUSP5) has been shown to play a key role in embryonic vascular development, but its role in post-ischemic angiogenesis is not known. We induced hind limb ischemia in mice and found robust upregulation of Dusp5 expression in ischemic hind limbs. Moreover, in vivo knockdown of Dusp5 resulted in impaired perfusion recovery in ischemic limbs and was associated with increased limb necrosis. In vitro studies showed upregulation of DUSP5 in human endothelial cells exposed to ischemia, and knockdown of DUSP5 in these ischemic endothelial cells resulted in impaired endothelial cell proliferation and angiogenesis, but did not alter apoptosis. Finally, we show that these effects of DUSP5 on post-ischemic angiogenesis are a result of DUSP5-dependent decrease in ERK1/2 phosphorylation and p21 protein expression. Thus, we have identified a role of DUSP5 in post-ischemic angiogenesis and implicated a DUSP5-ERK-p21 pathway that may serve as a therapeutic target for the modulation of post-ischemic angiogenesis in PAD.

Investigation of the mechanisms of cyclooxygenase-mediated mechanoreflex sensitization in a rat model of simulated peripheral artery disease.

Mechanical and metabolic stimuli within contracting skeletal muscles reflexly increase sympathetic nervous system activity and blood pressure. That reflex, termed the exercise pressor reflex, is exaggerated in patients with peripheral artery disease (PAD) and in a rat PAD model with a chronically ligated femoral artery. The cyclooxygenase (COX) pathway contributes to the exaggerated pressor response during rhythmic skeletal muscle contractions in patients with PAD, but the specific mechanism(s) of the COX-mediated exaggeration are not known. In decerebrate, unanesthetized rats with a chronically ligated femoral artery ("ligated" rats), we hypothesized that hindlimb arterial injection of the COX inhibitor indomethacin would reduce the pressor response during 1-Hz dynamic hindlimb skeletal muscle stretch; a model of the activation of the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex). In ligated rats (n = 7), indomethacin reduced the pressor response during stretch (control: 30 ± 4; indomethacin: 12 ± 3 mmHg; P < 0.01), whereas there was no effect in rats with "freely perfused" femoral arteries (n = 6, control: 18 ± 5; indomethacin: 17 ± 5 mmHg; P = 0.87). In ligated rats (n = 4), systemic indomethacin injection had no effect on the pressor response during stretch. Femoral artery ligation had no effect on skeletal muscle COX protein expression or activity or concentration of the COX metabolite prostaglandin E2. Conversely, femoral artery ligation increased expression of the COX metabolite receptors endoperoxide 4 and thromboxane A2-R in dorsal root ganglia tissue. We conclude that, in ligated rats, the COX pathway sensitizes the peripheral endings of mechanoreflex afferents, which occurs principally as a result of increased expression of COX metabolite receptors.NEW & NOTEWORTHY We demonstrate that the mechanoreflex is sensitized by the cyclooxygenase (COX) pathway within hindlimb skeletal muscles in the rat chronic femoral artery ligation model of simulated peripheral artery disease (PAD). The mechanism of sensitization appears attributable to increased receptors for COX metabolites on sensory neurons and not increased concentration of COX metabolites. Our data may carry important clinical implications for patients with PAD who demonstrate exaggerated increases in blood pressure during exercise compared with healthy counterparts.

Association of serum proprotein convertase subtilisin/kexin type 9 with early atherosclerosis in newly diagnosed type 2 diabetes mellitus.

BACKGROUND AND AIM: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is rapidly gaining attention as a potential risk of developing atherosclerosis due to its crucial role in the regulation of low-density lipoprotein cholesterol (LDL-C) metabolism. The present study investigated the relationship between serum PCSK9 levels and early atherosclerosis as assessed by carotid intimal-medial thickness (CIMT) and brachial-ankle pulse wave velocity (ba-PWV) in newly diagnosed type 2 diabetes mellitus (T2DM). METHODS AND RESULTS: A total of 100 newly diagnosed T2DM were enrolled and further divided into the thickened CIMT group (n = 41) and the non-thickened CIMT group (n = 59) according to the results of color Doppler ultrasonography. Serum PCSK9 levels, CIMT, ba-PWV, and metabolic parameters were measured. Patients in the thickened CIMT group had higher serum PCSK9 levels than patients in the non-thickened CIMT group (all P < 0.05). CIMT and ba-PWV were both positively correlated to serum PCSK9 levels, while serum PCSK9 levels were positively correlated to white blood cell count, neutrophil, lymphocyte, and high-sensitivity C-reactive protein (P < 0.05). Multiple linear regression indicated that serum PCSK9 level was an independent predictor of CIMT (ß = 0.637, P < 0.001) and ba-PWV (ß = 0.600, P < 0.001). Binary logistic regression analysis showed that serum PCSK9 levels were independent risk factors of thickened CIMT [OR = 1.120, 95%CI (1.041-1.204), P = 0.002]. CONCLUSION: Serum PCSK9 levels are significantly correlated with CIMT and ba-PWV, independent of CAD risk factors. Therefore, serum PCSK9 level may have the potential to serve as a prescriptive biomarker for early arteriosclerosis in newly diagnosed T2DM.

Identification of an Arginase II Inhibitor via RapidFire Mass Spectrometry Combined with Hydrophilic Interaction Chromatography.

Peripheral arterial disease (PAD) is an occlusive disease that can lead to atherosclerosis. The involvement of arginase II (Arg II) in PAD progression has been proposed. However, no promising drugs targeting Arg II have been developed to date for the treatment of PAD. In this study, we established a method for detecting the activity of Arg II via high-throughput label-free RapidFire mass spectrometry using hydrophilic interaction chromatography, which enables the direct measurement of l-ornithine produced by Arg II. This approach facilitated a robust high-concentration screening of fragment compounds and the identification of a fragment that inhibits the activity of Arg II. We further confirmed binding of the fragment to the potential allosteric site of Arg II using a surface plasmon resonance assay. We concluded that the identified fragment is a promising compound that may lead to novel drugs to treat PAD, and our method for detecting the activity of Arg II can be applied to large-scale high-throughput screening to identify other structural types of Arg II inhibitors.

Repetitive remote occlusion (RRO) stimulates eNOS-dependent blood flow and collateral expansion in hindlimb ischemia.

OBJECTIVE: Collateral expansion is an important compensatory mechanism to alleviate tissue ischemia after arterial occlusion. We investigated the efficacy and mechanisms of temporary remote hindlimb occlusion to stimulate contralateral blood flow and collateral expansion after hindlimb ischemia in mice and evaluated translation to peripheral artery disease in humans. METHODS AND RESULTS: We induced unilateral hindlimb ischemia via femoral artery excision in mice. We studied central hemodynamics, blood flow, and perfusion of the ischemic hindlimb during single and repetitive remote occlusion (RRO) of the contralateral non-ischemic hindlimb with a pressurized cuff. Similar experiments were performed in patients with unilateral peripheral artery disease (PAD). Contralateral occlusion of the non-ischemic hindlimb led to an acute increase in blood flow to the ischemic hindlimb without affecting central blood pressure and cardiac output. The increase in blood flow was sustained even after deflation of the pressure cuff. RRO over 12 days (8/day, each 5 min) led to significantly increased arterial inflow, lumen expansion of collateral arteries, and increased perfusion of the chronically ischemic hindlimb as compared to control. In NOS3-/- and after inhibition of NOS (L-NAME), and NO (ODQ), the acute and chronic effects of contralateral occlusion were abrogated and stimulation of guanylyl cyclase with cinaciguate exhibited a similar response as RRO and was not additive. Pilot studies in PAD patients demonstrated that contralateral occlusion increased arterial inflow to ischemic limbs and improved walking distance. CONCLUSIONS: Repetitive remote contralateral occlusion stimulates arterial inflow, perfusion, and functional collateral expansion in chronic hindlimb ischemia via an eNOS-dependent mechanism underscoring the potential of remote occlusion as a novel treatment option in peripheral artery disease.

The involvement of p38MAPK in the rat model of lower-extremity arterial ischemia-reperfusion injury.

OBJECTIVE: This study aims to investigate the regulatory role of p38 mitogen-activated protein kinase (p38MAPK) in rats with lower-extremity arterial ischemia-reperfusion injury. MATERIALS AND METHODS: A total of 60 Sprague-Dawley (SD) rats were randomly divided into four groups: control group (Group A), lower-extremity arterial ischemia-reperfusion group (Group B), lower-extremity arterial ischemic postconditioning group (Group C), and lower-extremity arterial ischemic postconditioning + SB203580 group (Group D, 5 µmol/L SB203580, the inhibitor of MAPK pathway, was injected after lower-extremity arterial ischemic postconditioning). The lower-extremity arterial vessels were collected after 24 h. The apoptosis in the lower-extremity arterial vessel in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The expression of phosphorylated (p)-p38MAPK was measured via Western blotting, and the level of p-activating transcription factor-2 (ATF-2) was detected via immunohistochemical method. RESULTS: The positive rate of apoptotic cells (%) in Group B was significantly increased compared to that in Group A (p<0.05). However, the positive rate was statistically decreased by postcondition in Group C, the rate was further reduced after injection of SB203580 in Group D compared to Group B (p<0.05). Compared with that in Group C, the expressions of p-p38MAPK and p-ATF-2 in Group D were significantly downregulated after injection of SB203580 (p<0.05). CONCLUSIONS: Lower-extremity arterial ischemia-reperfusion postconditioning can significantly reduce the apoptosis level in vascular tissues, decrease the expressions of p-p38MAPK and downstream factor ATF-2, and alleviate the damage in lower-extremity arterial vessels. The inhibition of MAPK pathway further restricted the apoptosis and contributed to a promoting role in the recovery of lower-extremity arterial ischemia-reperfusion injury.

Plasma Heme Oxygenase-1 Levels in Patients with Coronary and Peripheral Artery Diseases.

AIMS: Heme oxygenase-1 (HO-1) is an intracellular enzyme that catalyzes the oxidation of heme to generate CO, biliverdin, and iron. Since these products have antiatherogenic properties, HO-1 may play a protective role against the progression of atherosclerosis. However, plasma HO-1 levels in patients with atherosclerotic diseases, such as coronary artery disease (CAD) and peripheral artery disease (PAD), have not been clarified yet. METHODS: We investigated plasma HO-1 levels by ELISA in 410 consecutive patients undergoing elective coronary angiography who also had an ankle-brachial index (ABI) test for PAD screening. RESULTS: Of the 410 study patients, CAD was present in 225 patients (55%) (1-vessel (1-VD), n = 91; 2-vessel (2-VD), n = 66; 3-vessel disease (3-VD), n = 68). PAD (ABI < 0.9) was found in 36 (9%) patients. Plasma HO-1 levels did not differ between 225 patients with CAD and 185 without CAD (median 0.44 versus 0.35 ng/mL), but they were significantly lower in 36 patients with PAD than in 374 without PAD (0.27 versus 0.41 ng/mL, P < 0.02). After excluding the 36 patients with PAD, HO-1 levels were significantly higher in 192 patients with CAD than in 182 without CAD (0.45 versus 0.35 ng/mL, P < 0.05). HO-1 levels in 4 groups of CAD(-), 1-VD, 2-VD, and 3-VD were 0.35, 0.49, 0.44, and 0.44 ng/mL, respectively, and were highest in 1-VD (P < 0.05). In the multivariate analysis, HO-1 levels were inversely associated with PAD, whereas they were also associated with CAD. The odds ratios for PAD and CAD were 2.12 (95% CI = 1.03-4.37) and 0.65 (95% CI = 0.42-0.99) for the HO-1 level of <0.35 ng/mL, respectively. CONCLUSIONS: Plasma HO-1 levels were found to be low in patients with PAD, in contrast to high levels in patients with CAD.

Tollip Negatively Regulates Vascular Smooth Muscle Cell-Mediated Neointima Formation by Suppressing Akt-Dependent Signaling.

BACKGROUND: Tollip, a well-established endogenous modulator of Toll-like receptor signaling, is involved in cardiovascular diseases. The aim of this study was to investigate the role of Tollip in neointima formation and its associated mechanisms. METHODS AND RESULTS: In this study, transient increases in Tollip expression were observed in platelet-derived growth factor-BB-treated vascular smooth muscle cells and following vascular injury in mice. We then applied loss-of-function and gain-of-function approaches to elucidate the effects of Tollip on neointima formation. While exaggerated neointima formation was observed in Tollip-deficient murine neointima formation models, Tollip overexpression alleviated vascular injury-induced neointima formation by preventing vascular smooth muscle cell proliferation, dedifferentiation, and migration. Mechanistically, we demonstrated that Tollip overexpression may exert a protective role in the vasculature by suppressing Akt-dependent signaling, which was further confirmed in rescue experiments using the Akt-specific inhibitor (AKTI). CONCLUSIONS: Our findings indicate that Tollip protects against neointima formation by negatively regulating vascular smooth muscle cell proliferation, dedifferentiation, and migration in an Akt-dependent manner. Upregulation of Tollip may be a promising strategy for treating vascular remodeling-related diseases.

PCSK9 inhibition for LDL lowering and beyond - implications for patients with peripheral artery disease.

Low-density lipoprotein cholesterol (LDL-C) has been proven to be a causal factor of atherosclerosis and, along with other triggers like inflammation, the most frequent reason for peripheral arterial disease. Moreover, a linear correlation between LDL-C concentration and cardiovascular outcome in high-risk patients could be established during the past century. After the development of statins, numerous randomized trials have shown the superiority for LDL-C reduction and hence the decrease in cardiovascular outcomes including mortality. Over the past decades it became evident that more intense LDL-C lowering, by either the use of highly potent statin supplements or by additional cholesterol absorption inhibitor application, accounted for an even more profound cardiovascular risk reduction. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a serin protease with effect on the LDL receptor cycle leading to its degradation and therefore preventing continuing LDL-C clearance from the blood, is the target of a newly developed monoclonal antibody facilitating astounding LDL-C reduction far below to what has been set as target level by recent ESC/EAS guidelines in management of dyslipidaemias. Large randomized outcome trials including subjects with PAD so far have been able to prove significant and even more intense cardiovascular risk reduction via further LDL-C debasement on top of high-intensity statin medication. Another approach for LDL-C reduction is a silencing interfering RNA muting the translation of PCSK9 intracellularly. Moreover, PCSK9 concentrations are elevated in cells involved in plaque composition, so the potency of intracellular PCSK9 inhibition and therefore prevention or reversal of plaques may provide this mechanism of action on PCSK9 with additional beneficial effects on cells involved in plaque formation. Thus, simultaneous application of statins and PCSK9 inhibitors promise to reduce cardiovascular event burden by both LDL-C reduction and pleiotropic effects of both agents.

Phenotypic Consequences of a Genetic Predisposition to Enhanced Nitric Oxide Signaling.

BACKGROUND: Nitric oxide signaling plays a key role in the regulation of vascular tone and platelet activation. Here, we seek to understand the impact of a genetic predisposition to enhanced nitric oxide signaling on risk for cardiovascular diseases, thus informing the potential utility of pharmacological stimulation of the nitric oxide pathway as a therapeutic strategy. METHODS: We analyzed the association of common and rare genetic variants in 2 genes that mediate nitric oxide signaling (Nitric Oxide Synthase 3 [NOS3] and Guanylate Cyclase 1, Soluble, Alpha 3 [GUCY1A3]) with a range of human phenotypes. We selected 2 common variants (rs3918226 in NOS3 and rs7692387 in GUCY1A3) known to associate with increased NOS3 and GUCY1A3 expression and reduced mean arterial pressure, combined them into a genetic score, and standardized this exposure to a 5 mm Hg reduction in mean arterial pressure. Using individual-level data from 335 464 participants in the UK Biobank and summary association results from 7 large-scale genome-wide association studies, we examined the effect of this nitric oxide signaling score on cardiometabolic and other diseases. We also examined whether rare loss-of-function mutations in NOS3 and GUCY1A3 were associated with coronary heart disease using gene sequencing data from the Myocardial Infarction Genetics Consortium (n=27 815). RESULTS: A genetic predisposition to enhanced nitric oxide signaling was associated with reduced risks of coronary heart disease (odds ratio, 0.37; 95% confidence interval [CI], 0.31-0.45; P=5.5*10-26], peripheral arterial disease (odds ratio 0.42; 95% CI, 0.26-0.68; P=0.0005), and stroke (odds ratio, 0.53; 95% CI, 0.37-0.76; P=0.0006). In a mediation analysis, the effect of the genetic score on decreased coronary heart disease risk extended beyond its effect on blood pressure. Conversely, rare variants that inactivate the NOS3 or GUCY1A3 genes were associated with a 23 mm Hg higher systolic blood pressure (95% CI, 12-34; P=5.6*10-5) and a 3-fold higher risk of coronary heart disease (odds ratio, 3.03; 95% CI, 1.29-7.12; P=0.01). CONCLUSIONS: A genetic predisposition to enhanced nitric oxide signaling is associated with reduced risks of coronary heart disease, peripheral arterial disease, and stroke. Pharmacological stimulation of nitric oxide signaling may prove useful in the prevention or treatment of cardiovascular disease.

A Reliable Mouse Model of Hind limb Gangrene.

BACKGROUND: Lack of a reliable hind limb gangrene animal model limits preclinical studies of gangrene, a severe form of critical limb ischemia. We develop a novel mouse hind limb gangrene model to facilitate translational studies. METHODS: BALB/c, FVB, and C57BL/6 mice underwent femoral artery ligation (FAL) with or without administration of NG-nitro-L-arginine methyl ester (L-NAME), an endothelial nitric oxide synthase inhibitor. Gangrene was assessed using standardized ischemia scores ranging from 0 (no gangrene) to 12 (forefoot gangrene). Laser Doppler imaging (LDI) and DiI perfusion quantified hind limb reperfusion postoperatively. RESULTS: BALB/c develops gangrene with FAL-only (n = 11/11, 100% gangrene incidence), showing mean limb ischemia score of 12 on postoperative days (PODs) 7 and 14 with LDI ranging from 0.08 to 0.12 on respective PODs. Most FVB did not develop gangrene with FAL-only (n = 3/9, 33% gangrene incidence) but with FAL and L-NAME (n = 9/9, 100% gangrene incidence). Mean limb ischemia scores for FVB undergoing FAL with L-NAME were significantly higher than for FVB receiving FAL-only. LDI score and capillary density by POD 28 were significantly lower in FVB undergoing FAL with L-NAME. C57BL/6 did not develop gangrene with FAL-only or FAL and L-NAME. CONCLUSIONS: Reproducible murine gangrene models may elucidate molecular mechanisms for gangrene development, facilitating therapeutic intervention.

Lipoprotein-associated phospholipase A2 and risk of incident peripheral arterial disease: Findings from The Atherosclerosis Risk in Communities study (ARIC).

BACKGROUND AND AIMS: Results from prospective studies evaluating the relationship between elevated lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and incident peripheral arterial disease (PAD) have been mixed. We investigated whether higher Lp-PLA2 levels are associated with increased risk of incident PAD and whether PLA2G7 gene variants, which result in lower Lp-PLA2 levels, are associated with reduced risk of incident PAD. METHODS: Our analysis included 9922 participants (56% female; 21% African-American; mean age 63 years) without baseline PAD at ARIC Visit 4 (1996-1998), who had Lp-PLA2 activity measured and were subsequently followed for the development of PAD, defined by occurrence of a PAD-related hospitalization, through 2012. Cox proportional hazard models were performed to determine the association of Lp-PLA2 levels and PLA2G7 gene variants with incident PAD. RESULTS: During a median follow-up of 14.9 years, we identified 756 incident cases of PAD. In analyses adjusting for age, race, and sex, each standard deviation increment in Lp-PLA2 activity (62 nmol/ml/min) was associated with a higher risk of developing PAD (hazard ratio (HR) 1.17; 95% confidence interval (CI) 1.09, 1.26). This association remained significant after additional adjustment for risk factors, other cardiovascular disease, and medication use, but was strongly attenuated (HR: 1.09; 95% CI 1.00, 1.20). PLA2G7 variants were not associated with a lower risk of PAD in both white carriers (HR: 1.21; 95% CI: 0.17-8.56) and African-American carriers (HR: 0.83; 95% CI: 0.41-1.67), although statistical power was quite limited for this analysis, particularly in whites. CONCLUSIONS: While higher Lp-PLA2 activity was associated with an increased risk for incident PAD, it is likely a risk marker largely represented by traditional risk factors.

DNA Methyltransferase 1-Dependent DNA Hypermethylation Constrains Arteriogenesis by Augmenting Shear Stress Set Point.

BACKGROUND: Arteriogenesis is initiated by increased shear stress and is thought to continue until shear stress is returned to its original "set point." However, the molecular mechanism(s) through which shear stress set point is established by endothelial cells (ECs) are largely unstudied. Here, we tested the hypothesis that DNA methyltransferase 1 (DNMT1)-dependent EC DNA methylation affects arteriogenic capacity via adjustments to shear stress set point. METHODS AND RESULTS: In femoral artery ligation-operated C57BL/6 mice, collateral artery segments exposed to increased shear stress without a change in flow direction (ie, nonreversed flow) exhibited global DNA hypermethylation (increased 5-methylcytosine staining intensity) and constrained arteriogenesis (30% less diameter growth) when compared with segments exposed to both an increase in shear stress and reversed-flow direction. In vitro, ECs exposed to a flow waveform biomimetic of nonreversed collateral segments in vivo exhibited a 40% increase in DNMT1 expression, genome-wide hypermethylation of gene promoters, and a DNMT1-dependent 60% reduction in proarteriogenic monocyte adhesion compared with ECs exposed to a biomimetic reversed-flow waveform. These results led us to test whether DNMT1 regulates arteriogenic capacity in vivo. In femoral artery ligation-operated mice, DNMT1 inhibition rescued arteriogenic capacity and returned shear stress back to its original set point in nonreversed collateral segments. CONCLUSIONS: Increased shear stress without a change in flow direction initiates arteriogenic growth; however, it also elicits DNMT1-dependent EC DNA hypermethylation. In turn, this diminishes mechanosensing, augments shear stress set point, and constrains the ultimate arteriogenic capacity of the vessel. This epigenetic effect could impact both endogenous collateralization and treatment of arterial occlusive diseases.