The pro-resolving mediator, annexin A1 regulates blood pressure, and age-associated changes in cardiovascular function and remodeling.

Aging is associated with chronic, low-level inflammation which may contribute to cardiovascular pathologies such as hypertension and atherosclerosis. This chronic inflammation may be opposed by endogenous mechanisms to limit inflammation, for example, by the actions of annexin A1 (ANXA1), an endogenous glucocorticoid-regulated protein that has anti-inflammatory and pro-resolving activity. We hypothesized the pro-resolving mediator ANXA1 protects against age-induced changes in blood pressure (BP), cardiovascular structure and function, and cardiac senescence. BP was measured monthly in conscious mature (4-month) and middle-aged (12-month) ANXA1-deficient (ANXA1-/- ) and wild-type C57BL/6 mice. Body composition was measured using EchoMRI, and both cardiac and vascular function using ultrasound imaging. Cardiac hypertrophy, fibrosis and senescence, vascular fibrosis, elastin, and calcification were assessed histologically. Gene expression relevant to structural remodeling, inflammation, and cardiomyocyte senescence were also quantified. In C57BL/6 mice, progression from 4 to 12 months of age did not affect the majority of cardiovascular parameters measured, with the exception of mild cardiac hypertrophy, vascular calcium, and collagen deposition. Interestingly, ANXA1-/- mice exhibited higher BP, regardless of age. Additionally, age progression had a marked impact in ANXA1-/- mice, with markedly augmented vascular remodeling, impaired vascular distensibility, and body composition. Consistent with vascular dysfunction, cardiac dysfunction, and hypertrophy were also evident, together with markers of senescence and inflammation. These findings suggest that endogenous ANXA1 plays a critical role in regulating BP, cardiovascular function, and remodeling and delays cardiac senescence. Our findings support the development of novel ANXA1-based therapies to prevent age-related cardiovascular pathologies.

A high-sucrose diet exacerbates the left ventricular phenotype in a high fat-fed streptozotocin rat model of diabetic cardiomyopathy.

Left ventricular (LV) dysfunction is an early, clinically detectable sign of cardiomyopathy in type 2 diabetes mellitus (T2DM) that precedes the development of symptomatic heart failure. Preclinical models of diabetic cardiomyopathy are essential to develop therapies that may prevent or delay the progression of heart failure. This study examined the molecular, structural, and functional cardiac phenotype of two rat models of T2DM induced by a high-fat diet (HFD) with a moderate- or high-sucrose content (containing 88.9 or 346 g/kg sucrose, respectively), plus administration of low-dose streptozotocin (STZ). At 8 wk of age, male Sprague-Dawley rats commenced a moderate- or high-sucrose HFD. Two weeks later, rats received low-dose STZ (35 mg/kg ip for 2 days) and remained on their respective diets. LV function was assessed by echocardiography 1 wk before end point. At 22 wk of age, blood and tissues were collected postmortem. Relative to chow-fed sham rats, diabetic rats on a moderate- or high-sucrose HFD displayed cardiac reactive oxygen species dysregulation, perivascular fibrosis, and impaired LV diastolic function. The diabetes-induced impact on LV adverse remodeling and diastolic dysfunction was more apparent when a high-sucrose HFD was superimposed on STZ. In conclusion, a high-sucrose HFD in combination with low-dose STZ produced a cardiac phenotype that more closely resembled T2DM-induced cardiomyopathy than STZ diabetic rats subjected to a moderate-sucrose HFD.NEW & NOTEWORTHY Left ventricular dysfunction and adverse remodeling were more pronounced in diabetic rats that received low-dose streptozotocin (STZ) and a high-sucrose high-fat diet (HFD) compared with those on a moderate-sucrose HFD in combination with STZ. Our findings highlight the importance of sucrose content in diet composition, particularly in preclinical studies of diabetic cardiomyopathy, and demonstrate that low-dose STZ combined with a high-sucrose HFD is an appropriate rodent model of cardiomyopathy in type 2 diabetes.

Shear stress sensitizes TRPV4 in endothelium-dependent vasodilatation.

The aim of this study was to better understand the role of TRPV4 in the regulation of blood vessel dilatation by blood flow and activation of GPCRs. Using pressure myography, the dilator responses to the TRPV4 agonist GSK1016790A and to acetylcholine, were examined in rat cremaster arterioles exposed to either no shear stress or to 200 µl/min flow for 6 min. In control vessels GSK1016709A caused vasodilatation (pEC50 7.73 ±â€¯0.12 M, ΔDmax 97 ±â€¯3%) which was significantly attenuated by the TRPV4 antagonists GSK2193874 (100 nM) (pEC50 6.19 ±â€¯0.11 M, p < 0.05) and HC067047 (300 nM) (pEC50 6.44 ±â€¯0.12 M) and abolished by removal of the endothelium. Shear conditioned arterioles were significantly more sensitive to GSK1016790A (pEC50 8.34 ±â€¯0.11, p < 0.05). Acetylcholine-induced vasodilatation (pEC50 7.02 ±â€¯0.07 M, ΔDmax 93 ±â€¯2%) was not affected by shear forces (pEC50 7.08 ±â€¯0.07 M, ΔDmax 95 ±â€¯1%). The dilator response to acetylcholine was unaffected by the TRPV4 antagonist GSK2193874 in control arterioles (pEC50 7.24 ±â€¯0.07 M, ΔDmax 97 ±â€¯2%). However, in shear treated arterioles, the acetylcholine-response was significantly attenuated by GSK2193874 (pEC50 6.25 ±â€¯0.12 M, p < 0.05) indicating an induced interaction between TRPV4 and muscarinic receptors. TRPV4 antibodies localized TRPV4 to the endothelium and shear stress had no effect on its localisation. Finally, agonist activation of the M3 muscarinic receptor opened TRPV4 in HEK293 cells. We concluded that shear stress increases endothelial TRPV4 agonist sensitivity and links TRPV4 activation to muscarinic receptor mediated endothelium-dependent vasodilatation, providing strong evidence that blood flow modulates downstream signalling from at least one but not all GPCRs expressed in the endothelium.

The HNO donor Angeli's salt offers potential haemodynamic advantages over NO or dobutamine in ischaemia-reperfusion injury in the rat heart ex vivo.

Available inotropic pharmacotherapy for acute heart failure (HF) remains largely ineffective at ameliorating marked impairments in contractile function. Nitroxyl (HNO), the redox sibling of NO•, has recently attracted interest as a therapeutic approach for acute HF. We now compare the impact of ischaemia-reperfusion (I-R) injury on acute haemodynamic responsiveness of the HNO donor, Angeli's salt (AS), to that of NO and dobutamine. Dose-response curves to bolus doses of AS, diethylamine NONOate (DEA/NO, both 0.001-µmol) and dobutamine (0.1-100 nmol) were performed in rat isolated hearts, following I-R or normoxic perfusion. An additional 10µmol dose of Angeli's salt was included, to permit roughly equivalent inotropic responses to dobutamine. Changes in cardiac contraction, heart rate and coronary flow (CF) were determined. Although AS and DEA/NO elicited comparable dose-dependent increases in CF in normoxic hearts, only AS vasodilation was preserved after I-R. AS and dobutamine elicited dose-dependent inotropic responses in normoxic hearts and I-R blunted inotropic responses to both. Dobutamine however increased heart rate, which was exacerbated by I-R; this was not evident with AS. Further, AS infusion during reperfusion (1µM), in a separate cohort of rat hearts, improved recovery of cardiac contractility, with lower incidence of I-R-induced ventricular fibrillation. In conclusion, these observations suggest that HNO offers haemodynamic advantages over NO following I-R. Although I-R suppresses inotropy to both agents, residual contractile responses to AS following I-R is likely free of concomitant pro-arrhythmic events. HNO donors may thus offer haemodynamic advantages over existing pharmacotherapy in acute HF.

Flavonols in the Prevention of Diabetes-induced Vascular Dysfunction.

As flavonols are present in fruits and vegetables, they are consumed in considerable amounts in the diet. There is growing evidence that the well-recognized antioxidant, anti-inflammatory, and vasorelaxant actions of flavonols may, at least in part, result from modulation of biochemical signaling pathways and kinases. It is well established that diabetes is associated with increased cardiovascular morbidity and mortality. Despite clinical management of blood glucose levels, diabetes often results in cardiovascular disease. There is good evidence that endothelial dysfunction contributes significantly to the progression of diabetic cardiovascular diseases. This review describes the biological actions of flavonols that may ameliorate adverse cardiovascular events in diabetes. We discuss evidence that flavonols may be developed as novel pharmacological agents to prevent diabetes-induced vascular dysfunction.

Cardioprotective 3',4'-dihydroxyflavonol attenuation of JNK and p38(MAPK) signalling involves CaMKII inhibition.

DiOHF (3',4'-dihydroxyflavonol) is cardioprotective against I/R (ischaemia/reperfusion) injury. The biological activities of flavonols are associated with kinase modulation to alter cell signalling. We thus investigated the effects of DiOHF on the activation of MAPKs (mitogen-activated protein kinases) that regulate the cardiac stress response. In an ovine model of I/R, JNK (c-Jun N-terminal kinase), p38(MAPK), ERK (extracellular-signal-regulated kinase) and Akt were activated, and NP202, a pro-drug of DiOHF, reduced infarct size and inhibited JNK and p38(MAPK) activation, whereas ERK and Akt phosphorylation were unaltered. Similarly, in cultured myoblasts, DiOHF pre-treatment preserved viability and inhibited activation of JNK and p38(MAPK), but not ERK in response to acute oxidative and chemotoxic stress. Furthermore, DiOHF prevented stress-activation of the direct upstream regulators MKK4/7 (MAPK kinase 4/7) and MKK3/6 respectively. We utilized small-molecule affinity purification and identified CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) as a kinase targeted by DiOHF and demonstrated potent CaMKII inhibition by DiOHF in vitro. Moreover, the specific inhibition of CaMKII with KN-93, but not KN-92, prevented oxidative stress-induced activation of JNK and p38(MAPK). The present study indicates DiOHF inhibition of CaMKII and attenuation of MKK3/6→p38(MAPK) and MKK4/7→JNK signalling as a requirement for the protective effects of DiOHF against stress stimuli and myocardial I/R injury.

Formyl peptide receptor 1 mitigates colon inflammation and maintains mucosal homeostasis through the inhibition of CREB-C/EBPß-S100a8 signaling.

Excessive inflammatory responses are the main characteristic of ulcerative colitis (UC). Activation of formyl peptide receptor 1 (FPR1) has been found to promote the proliferation and migration of epithelial cells, but its role and therapeutic potential in UC remain unclear. This study observed an increased expression of FPR1 in a mouse model of colitis. Interestingly, FPR1 deficiency exacerbated UC and increased the secretion of the proinflammatory mediator from immune cells (e.g. macrophages), S100a8, a member of the damage-associated molecular patterns. Notably, the administration of the FPR agonist Cmpd43 ameliorated colon injury in a preclinical mice model of UC, likely via inhibiting phosphorylation of cyclic adenosine monophosphate-response element-binding protein and expression of CCAAT/enhancer-binding protein ß, which in turn suppressed the secretion of S100a8. In conclusion, these findings discovered a novel role of FPR1 in the development of colitis and will facilitate the development of FPR1-based pharmacotherapy to treat UC.

Novel formylpeptide receptor 1/2 agonist limits hypertension-induced cardiovascular damage.

AIMS: Formylpeptide receptors (FPRs) play a critical role in the regulation of inflammation, an important driver of hypertension-induced end-organ damage. We have previously reported that the biased FPR small-molecule agonist, compound17b (Cmpd17b), is cardioprotective against acute, severe inflammatory insults. Here, we reveal the first compelling evidence of the therapeutic potential of this novel FPR agonist against a longer-term, sustained inflammatory insult, i.e. hypertension-induced end-organ damage. The parallels between the murine and human hypertensive proteome were also investigated. METHODS AND RESULTS: The hypertensive response to angiotensin II (Ang II, 0.7 mg/kg/day, s.c.) was attenuated by Cmpd17b (50 mg/kg/day, i.p.). Impairments in cardiac and vascular function assessed via echocardiography were improved by Cmpd17b in hypertensive mice. This functional improvement was accompanied by reduced cardiac and aortic fibrosis and vascular calcification. Cmpd17b also attenuated Ang II-induced increased cardiac mitochondrial complex 2 respiration. Proteomic profiling of cardiac and aortic tissues and cells, using label-free nano-liquid chromatography with high-sensitivity mass spectrometry, detected and quantified ∼6000 proteins. We report hypertension-impacted protein clusters associated with dysregulation of inflammatory, mitochondrial, and calcium responses, as well as modified networks associated with cardiovascular remodelling, contractility, and structural/cytoskeletal organization. Cmpd17b attenuated hypertension-induced dysregulation of multiple proteins in mice, and of these, ∼110 proteins were identified as similarly dysregulated in humans suffering from adverse aortic remodelling and cardiac hypertrophy. CONCLUSION: We have demonstrated, for the first time, that the FPR agonist Cmpd17b powerfully limits hypertension-induced end-organ damage, consistent with proteome networks, supporting development of pro-resolution FPR-based therapeutics for treatment of systemic hypertension complications.

Low intrinsic exercise capacity in rats predisposes to age-dependent cardiac remodeling independent of macrovascular function.

Rats selectively bred for low (LCR) or high (HCR) intrinsic running capacity simultaneously present with contrasting risk factors for cardiovascular and metabolic disease. However, the impact of these phenotypes on left ventricular (LV) morphology and microvascular function, and their progression with aging, remains unresolved. We tested the hypothesis that the LCR phenotype induces progressive age-dependent LV remodeling and impairments in microvascular function, glucose utilization, and ß-adrenergic responsiveness, compared with HCR. Hearts and vessels isolated from female LCR (n = 22) or HCR (n = 26) were studied at 12 and 35 wk. Nonselected N:NIH founder rats (11 wk) were also investigated (n = 12). LCR had impaired glucose tolerance and elevated plasma insulin (but not glucose) and body-mass at 12 wk compared with HCR, with early LV remodeling. By 35 wk, LV prohypertrophic and glucose transporter GLUT4 gene expression were up- and downregulated, respectively. No differences in LV ß-adrenoceptor expression or cAMP content between phenotypes were observed. Macrovascular endothelial function was predominantly nitric oxide (NO)-mediated in both phenotypes and remained intact in LCR for both age-groups. In contrast, mesenteric arteries microvascular endothelial function, which was impaired in LCR rats regardless of age. At 35 wk, endothelial-derived hyperpolarizing factor-mediated relaxation was impaired whereas the NO contribution to relaxation is intact. Furthermore, there was reduced ß2-adrenoceptor responsiveness in both aorta and mesenteric LCR arteries. In conclusion, diminished intrinsic exercise capacity impairs systemic glucose tolerance and is accompanied by progressive development of LV remodeling. Impaired microvascular perfusion is a likely contributing factor to the cardiac phenotype.

Inhibition of platelet-mediated arterial thrombosis and platelet granule exocytosis by 3',4'-dihydroxyflavonol and quercetin.

Flavonols are polyphenolic compounds with broad-spectrum kinase inhibitory, as well as potent anti-oxidant and anti-inflammatory properties. Anti-platelet potential of quercetin (Que) and several related flavonoids have been reported; however, few studies have assessed the ability of flavonols to inhibit exocytosis of different platelet granules or to inhibit thrombus formation in vivo. 3',4'-Dihydroxyflavonol (DiOHF) is a flavonol which is structurally related to Que and has been shown to have greater anti-oxidant capacity and to improve the endothelial function in the context of diabetes and ischaemia/reperfusion injury. While the structural similarity to Que suggests DiOHF may have a potential to inhibit platelet function, no studies have assessed the anti-platelet potential of DiOHF. We therefore investigated platelet granule inhibition and potential to delay arterial thrombosis by Que and DiOHF. Both Que and DiOHF showed inhibition of collagen, adenosine diphosphate and arachidonic acid stimulated platelet aggregation, agonist-induced GPIIb/IIIa activation as demonstrated by PAC-1 and fibrinogen binding. While both flavonols inhibited agonist-induced granule exocytosis, greater inhibition of dense granule exocytosis occurred with DiOHF as measured by both ATP release and flow cytometry. In contrast, while Que inhibited agonist-induced P-selectin expression, as measured by both platelet surface P-selectin expression and upregulation of surface GPIIIa expression, inhibition by DiOHF was not significant for either parameter. C57BL/6 mice treated with 6 mg kg(-1) IV Que or DiOHF maintained greater blood flow following FeCl3-induced carotid artery injury when compared to the vehicle control. We provide evidence that Que and DiOHF improve blood flow following arterial injury in part by attenuating platelet granule exocytosis.

Endothelium-dependent relaxation is impaired in Schlager hypertensive (BPH/2J) mice by region-specific mechanisms in conductance and resistance arteries.

AIMS: Endothelial dysfunction and arterial stiffness are hallmarks of hypertension, and major risk factors for cardiovascular disease. BPH/2J (Schlager) mice are a genetic model of spontaneous hypertension, but little is known about the vascular pathophysiology of these mice and the region-specific differences between vascular beds. Therefore, this study compared the vascular function and structure of large conductance (aorta and femoral) and resistance (mesenteric) arteries of BPH/2J mice with their normotensive BPN/2J counterparts. MAIN METHODS: Blood pressure was measured in BPH/2J and BPN/3J mice via pre-implanted radiotelemetry probes. At endpoint, vascular function and passive mechanical wall properties were assessed using wire and pressure myography, qPCR and histology. KEY FINDINGS: Mean arterial blood pressure was elevated in BPH/2J mice compared to BPN/3J controls. Endothelium-dependent relaxation to acetylcholine was attenuated in both the aorta and mesenteric arteries of BPH/2J mice, but through different mechanisms. In the aorta, hypertension reduced the contribution of prostanoids. Conversely, in the mesenteric arteries, hypertension reduced the contribution of both nitric oxide and endothelium-dependent hyperpolarization. Hypertension reduced volume compliance in both femoral and mesenteric arteries, but hypertrophic inward remodelling was only observed in the mesenteric arteries of BPH/2J mice. SIGNIFICANCE: This is the first comprehensive investigation of vascular function and structural remodelling in BPH/2J mice. Overall, hypertensive BPH/2J mice exhibited endothelial dysfunction and adverse vascular remodelling in the macro- and microvasculature, underpinned by distinct region-specific mechanisms. This highlights BPH/2J mice as a highly suitable model for evaluating novel therapeutics to treat hypertension-associated vascular dysfunction.

The small-molecule formyl peptide receptor biased agonist, compound 17b, is a vasodilator and anti-inflammatory in mouse precision-cut lung slices.

BACKGROUND AND PURPOSE: Pulmonary arterial hypertension (PAH), a rare fatal disorder characterised by inflammation, vascular remodelling and vasoconstriction. Current vasodilator therapies reduce pulmonary arterial pressure but not mortality. The G-protein coupled formyl peptide receptors (FPRs) mediates vasodilatation and resolution of inflammation, actions possibly beneficial in PAH. We investigated dilator and anti-inflammatory effects of the FPR biased agonist compound 17b in pulmonary vasculature using mouse precision-cut lung slices (PCLS). EXPERIMENTAL APPROACH: PCLS from 8-week-old male and female C57BL/6 mice, intrapulmonary arteries were pre-contracted with 5-HT for concentration-response curves to compound 17b and 43, and standard-of-care drugs, sildenafil, iloprost and riociguat. Compound 17b-mediated relaxation was assessed with FPR antagonists or inhibitors and in PCLS treated with TNF-α or LPS. Cytokine release from TNF-α- or LPS-treated PCLS ± compound 17b was measured. KEY RESULTS: Compound 17b elicited concentration-dependent vasodilation, with potencies of iloprost > compound 17b = riociguat > compound 43 = sildenafil. Compound 17b was inhibited by the FPR1 antagonist cyclosporin H but not by soluble guanylate cyclase, nitric oxide synthase or cyclooxygenase inhibitors. Under inflammatory conditions, the efficacy and potency of compound 17b were maintained, while iloprost and sildenafil were less effective. Additionally, compound 17b inhibited secretion of PAH-relevant cytokines via FPR2. CONCLUSIONS AND IMPLICATIONS: Vasodilation to compound 17b but not standard-of-care vasodilators, is maintained under inflammatory conditions, with additional inhibition of PAH-relevant cytokine release. This provides the first evidence that targeting FPR, with biased agonist, simultaneously targets vascular function and inflammation, supporting the development of FPR-based pharmacotherapy to treat PAH.

2-Morpholinoisoflav-3-enes as flexible intermediates in the synthesis of phenoxodiol, isophenoxodiol, equol and analogues: vasorelaxant properties, estrogen receptor binding and Rho/RhoA kinase pathway inhibition.

Isoflavone consumption correlates with reduced rates of cardiovascular disease. Epidemiological studies and clinical data provide evidence that isoflavone metabolites, such as the isoflavan equol, contribute to these beneficial effects. In this study we developed a new route to isoflavans and isoflavenes via 2-morpholinoisoflavenes derived from a condensation reaction of phenylacetaldehydes, salicylaldehydes and morpholine. We report the synthesis of the isoflavans equol and deoxygenated analogues, and the isoflavenes 7,4'-dihydroxyisoflav-3-ene (phenoxodiol, haganin E) and 7,4'-dihydroxyisoflav-2-ene (isophenoxodiol). Vascular pharmacology studies reveal that all oxygenated isoflavans and isoflavenes can attenuate phenylephrine-induced vasoconstriction, which was unaffected by the estrogen receptor antagonist ICI 182,780. Furthermore, the compounds inhibited U46619 (a thromboxane A(2) analogue) induced vasoconstriction in endothelium-denuded rat aortae, and reduced the formation of GTP RhoA, with the effects being greatest for equol and phenoxodiol. Ligand displacement studies of rat uterine cytosol estrogen receptor revealed the compounds to be generally weak binders. These data are consistent with the vasorelaxation activity of equol and phenoxodiol deriving at least in part by inhibition of the RhoA/Rho-kinase pathway, and along with the limited estrogen receptor affinity supports a role for equol and phenoxodiol as useful agents for maintaining cardiovascular function with limited estrogenic effects.

Novel strategies to promote resolution of inflammation to treat lower extremity artery disease.

Lower extremity artery disease (LEAD) is a chronic inflammatory disease that occurs when atherosclerotic plaques form in the lower extremities, which may lead to amputation if not manged properly. Given clinical standardcare (pharmacological and surgical) have limited efficacy in LEAD, developing novel strategies to manage LEAD remains an unmet clinical need. Given that active resolution of inflammation is essential to facilitate tissue healing and repair, failure to resolve inflammation may lead to chronic inflammation, dysregulated cellular homeostasis and adverse tissue remodeling. Several studies have demonstrated the importance of the balance between endogenous pro-resolving mediators and pro-inflammatory factors. There is growing evidence to suggest endogenous pro-resolving mediators engage with pro-resolving G-protein-coupled receptors to reduce the initiation and progression of inflammatory responses and to increase therapeutic angiogenesis in LEAD. Here, we highlight the mechanisms and the consequences of resolved inflammation, and the therapeutic potential of endogenous pro-resolving mediators-based strategy for this devastating disease.

NP202 treatment improves left ventricular systolic function and attenuates pathological remodelling following chronic myocardial infarction.

AIMS: Myocardial injury is a major contributor to left ventricular (LV) remodelling activating neurohormonal and inflammatory processes that create an environment of enhanced oxidative stress. This results in geometric and structural alterations leading to reduced LV systolic function. In this study we evaluated the efficacy of NP202, a synthetic flavonol, on cardiac remodelling in a chronic model of myocardial infarction (MI). MAIN METHODS: A rat model of chronic MI was induced by permanent surgical ligation of the coronary artery. NP202 treatment was commenced 2 days post-MI for 6 weeks at different doses (1, 10 and 20 mg/kg/day) to determine efficacy. Cardiac function was assessed by echocardiography prior to treatment and at week 6, and pressure-volume measurements were performed prior to tissue collection. Tissues were analysed for changes in fibrotic and inflammatory markers using immunohistochemistry and gene expression analysis. KEY FINDINGS: Rats treated with NP202 demonstrated improved LV systolic function and LV geometry compared to vehicle treated animals. Furthermore, measures of hypertrophy and interstitial fibrosis were attenuated in the non-infarct region of the myocardium with NP202 at the higher dose of 20 mg/kg (P < 0.05). At the tissue level, NP202 reduced monocyte chemoattractant protein-1 expression (P < 0.05) and tended to attenuate active caspase-3 expression to similar levels observed in sham animals (P = 0.075). SIGNIFICANCE: Improved LV function and structural changes observed with NP202 may be mediated through inhibition of inflammatory and apoptotic processes in the MI setting. NP202 could therefore prove a useful addition to standard therapy in patients with post-MI LV dysfunction.

Cardioprotective actions of nitroxyl donor Angeli's salt are preserved in the diabetic heart and vasculature in the face of nitric oxide resistance.

BACKGROUND AND PURPOSE: The risk of fatal cardiovascular events is increased in patients with type 2 diabetes mellitus (T2DM). A major contributor to poor prognosis is impaired nitric oxide (NO•) signalling at the level of tissue responsiveness, termed NO• resistance. This study aimed to determine if T2DM promotes NO• resistance in the heart and vasculature and whether tissue responsiveness to nitroxyl (HNO) is affected. EXPERIMENTAL APPROACH: At 8 weeks of age, male Sprague-Dawley rats commenced a high-fat diet. After 2 weeks, the rats received low-dose streptozotocin (two intraperitoneal injections, 35 mg·kg-1 , over two consecutive days) and continued on the same diet. Twelve weeks later, isolated hearts were Langendorff-perfused to assess responses to the NO• donor diethylamine NONOate (DEA/NO) and the HNO donor Angeli's salt. Isolated mesenteric arteries were utilised to measure vascular responsiveness to the NO• donors sodium nitroprusside (SNP) and DEA/NO, and the HNO donor Angeli's salt. KEY RESULTS: Inotropic, lusitropic and coronary vasodilator responses to DEA/NO were impaired in T2DM hearts, whereas responses to Angeli's salt were preserved or enhanced. Vasorelaxation to Angeli's salt was augmented in T2DM mesenteric arteries, which were hyporesponsive to the relaxant effects of SNP and DEA/NO. CONCLUSION AND IMPLICATIONS: This is the first evidence that inotropic and lusitropic responses are preserved, and NO• resistance in the coronary and mesenteric vasculature is circumvented, by the HNO donor Angeli's salt in T2DM. These findings highlight the cardiovascular therapeutic potential of HNO donors, especially in emergencies such as acute ischaemia or heart failure.

Modulation of the glucagon-like peptide-1 receptor signaling by naturally occurring and synthetic flavonoids.

The glucagon-like peptide 1 receptor (GLP-1R) is a promising target for the treatment of type II diabetes mellitus because of its role in metabolic homeostasis. In recent years, difficulties with peptide therapies have driven the search for small-molecule compounds to modulate the activity of this receptor. We recently identified quercetin, a naturally occurring flavonoid, as a probe-dependent, pathway-selective allosteric modulator of GLP-1R-mediated signaling. Using Chinese hamster ovary cells expressing the human GLP-1R, we have now extended this work to identify the structural requirements of flavonoids to modify GLP-1R binding and signaling (cAMP formation and intracellular Ca(2+) mobilization) of each of the GLP-1R endogenous agonists, as well as the clinically used exogenous peptide mimetic exendin-4. This study identified a chemical series of hydroxyl flavonols with the ability to selectively augment calcium (Ca(2+)) signaling in a peptide agonist-specific manner, with effects only on truncated GLP-1 peptides [GLP-1(7-36)NH(2) and GLP-1(7-37)] and exendin-4, but not on oxyntomodulin or full-length GLP-1 peptides [GLP-1(1-36)NH(2) and GLP-1(1-37)]. In addition, the 3-hydroxyl group on the flavone backbone (i.e., a flavonol) was essential for this activity, however insufficient on its own, to produce the allosteric effects. In contrast to hydroxyl flavonols, catechin had no effect on peptide-mediated Ca(2+) signaling but negatively modulated peptide-mediated cAMP formation in a probe-dependent manner. These data represent a detailed examination of the action of different flavonoids on peptide agonists at the GLP-1R and may aid in the development of future small molecule compounds targeted at this receptor.

Synthesis of a hypoxia-targeted conjugate of the cardioprotective agent 3',4'-dihydroxyflavonol and evaluation of its ability to reduce ischaemia/reperfusion injury.

3',4'-Dihydroxyflavonol (DiOHF) is a cardioprotective flavonol that reduces injury associated with myocardial ischaemia and reperfusion. We hypothesized that the efficacy of DiOHF could be enhanced through its targeting to hypoxic regions of partial reperfusion. Copper(I)-catalyzed ligation of an azide-modified DiOHF analogue to 2-propargyl-nitroimidazole afforded a DiOHF-nitroimidazole conjugate (DiOHF-NIm). When incubated with Con8 cells under normoxic conditions DiOHF-NIm could be detected in both the culture supernatant and cell lysate, whereas under hypoxic conditions it was present in substantially reduced amounts consistent with its selective metabolism under hypoxia. DiOHF-NIm possessed antioxidant activity comparable to DiOHF through scavenging of superoxide produced by NADPH/NADPH oxidase, but had significantly attenuated vasorelaxant activity. DiOHF-NIm treatment significantly reduced lactate dehydrogenase release following ischaemia/reperfusion in hindlimbs of anaesthetized rats (p <0.05), to a level similar to DiOHF treatment but also at earlier time points. DiOHF-NIm significantly reduced levels of myeloperoxidase (p <0.05), a biomarker of neutrophil accumulation, whereas the reduction afforded by DiOHF was not significant. DiOHF-NIm therefore represents a promising potential therapeutic for ischaemia/reperfusion injury.

Allosteric ligands of the glucagon-like peptide 1 receptor (GLP-1R) differentially modulate endogenous and exogenous peptide responses in a pathway-selective manner: implications for drug screening.

The glucagon-like peptide-1 (GLP-1) receptor is a key regulator of insulin secretion and a major therapeutic target for treatment of diabetes. However, GLP-1 receptor function is complex, with multiple endogenous peptides that can interact with the receptor, including full-length (1-37) and truncated (7-37) forms of GLP-1 that can each exist in an amidated form and the related peptide oxyntomodulin. We have investigated two GLP-1 receptor allosteric modulators, Novo Nordisk compound 2 (6,7-dichloro2-methylsulfonyl-3-tert-butylaminoquinoxaline) and quercetin, and their ability to modify binding and signaling (cAMP formation, intracellular Ca(2+) mobilization, and extracellular signal-regulated kinase 1/2 phosphorylation) of each of the naturally occurring endogenous peptide agonists, as well as the clinically used peptide mimetic exendin-4. We identified and quantified stimulus bias across multiple endogenous peptides, with response profiles for truncated GLP-1 peptides distinct from those of either the full-length GLP-1 peptides or oxyntomodulin, the first demonstration of such behavior at the GLP-1 receptor. Compound 2 selectively augmented cAMP signaling but did so in a peptide-agonist dependent manner having greatest effect on oxyntomodulin, weaker effect on truncated GLP-1 peptides, and negligible effect on other peptide responses; these effects were principally driven by parallel changes in peptide agonist affinity. In contrast, quercetin selectively modulated calcium signaling but with effects only on truncated GLP-1 peptides or exendin and not oxyntomodulin or full-length peptides. These data have significant implications for how GLP-1 receptor targeted drugs are screened and developed, whereas the allosterically driven, agonist-selective, stimulus bias highlights the potential for distinct clinical efficacy depending on the properties of individual drugs.

Effects of 3',4'-dihydroxyflavonol on vascular contractions of rat aortic rings.

1. 3',4'-Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti-oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions. 2. Contractile and relaxation responses were determined in rat endothelium-denuded aortic rings mounted in organ baths. In addition, the phosphorylation of myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17) was determined using western blot analysis. Levels of GTP RhoA, as a marker of RhoA activation, were also measured. 3. Cumulative addition of increasing concentrations of NaF (3.0-12.0 mmol/L) or U46619 (1.0-1000 nmol/L) concentration-dependently increased vascular tension. These responses were inhibited by pretreatment of aortic rings with DiOHF (10, 30 or 100 micromol/L), which dose-dependently decreased vascular contractions induced by 8.0 mmol/L NaF, 30 nmol/L U46619, 0.1 micromol/L phenylephrine and 50 mmol/L KCl. 4. The K(+) channel blockers 4-aminopyridine (3 mmol/L), charybdotoxin (10 nmol/L), apamin (500 nmol/L) and glibenclamide (10 micromol/L) had no effect on vascular relaxation induced by DiOHF (1-30 micromol/L). 5. At 30 micromol/L, DiOHF decreased the activation of RhoA and subsequent phosphorylation of MYPT1, CPI-17 and MLC(20) to almost basal levels. 6. In conclusion, DiOHF decreases vascular contraction at least partly by inhibition of the RhoA/Rho-kinase pathway in rat endothelium-denuded aorta. These results suggest that DiOHF may have therapeutic potential in the treatment of cardiovascular diseases.