Selective Phosphodiesterase 1 Inhibitor BTTQ Reduces Blood Pressure in Spontaneously Hypertensive and Dahl Salt Sensitive Rats: Role of Peripheral Vasodilation.

Regulation of the peripheral vascular resistance via modulating the vessel diameter has been considered as a main determinant of the arterial blood pressure. Phosphodiesterase enzymes (PDE1-11) hydrolyse cyclic nucleotides, which are key players controlling the vessel diameter and, thus, peripheral resistance. Here, we have tested and reported the effects of a novel selective PDE1 inhibitor (BTTQ) on the cardiovascular system. Normal Sprague Dawley, spontaneously hypertensive (SHR), and Dahl salt-sensitive rats were used to test in vivo the efficacy of the compound. Phosphodiesterase radiometric enzyme assay revealed that BTTQ inhibited all three isoforms of PDE1 in nanomolar concentration, while micromolar concentrations were needed to induce effective inhibition for other PDEs. The myography study conducted on mesenteric arteries revealed a potent vasodilatory effect of the drug, which was confirmed in vivo by an increase in the blood flow in the rat ear arteriols reflected by the rise in the temperature. Furthermore, BTTQ proved a high efficacy in lowering the blood pressure about 9, 36, and 24 mmHg in normal Sprague Dawley, SHR and, Dahl salt-sensitive rats, respectively, compared to the vehicle-treated group. Moreover, additional blood pressure lowering of about 22 mmHg could be achieved when BTTQ was administered on top of ACE inhibitor lisinopril, a current standard of care in the treatment of hypertension. Therefore, PDE1 inhibition induced efficient vasodilation that was accompanied by a significant reduction of blood pressure in different hypertensive rat models. Administration of BTTQ was also associated with increased heart rate in both models of hypertension as well as in the normotensive rats. Thus, PDE1 appears to be an attractive therapeutic target for the treatment of resistant hypertension, while tachycardia needs to be addressed by further compound structural optimization.

PDE1 inhibition facilitates proteasomal degradation of misfolded proteins and protects against cardiac proteinopathy.

No current treatment targets cardiac proteotoxicity or can reduce mortality of heart failure (HF) with preserved ejection fraction (HFpEF). Selective degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is vital to the cell. Proteasome impairment contributes to HF. Activation of cAMP-dependent protein kinase (PKA) or cGMP-dependent protein kinase (PKG) facilitates proteasome functioning. Phosphodiesterase 1 (PDE1) hydrolyzes both cyclic nucleotides and accounts for most PDE activities in human myocardium. We report that PDE1 inhibition (IC86430) increases myocardial 26S proteasome activities and UPS proteolytic function in mice. Mice with CryABR120G-based proteinopathy develop HFpEF and show increased myocardial PDE1A expression. PDE1 inhibition markedly attenuates HFpEF, improves mouse survival, increases PKA-mediated proteasome phosphorylation, and reduces myocardial misfolded CryAB. Therefore, PDE1 inhibition induces PKA- and PKG-mediated promotion of proteasomal degradation of misfolded proteins and treats HFpEF caused by CryABR120G, representing a potentially new therapeutic strategy for HFpEF and heart disease with increased proteotoxic stress.

Factors associated with stroke, myocardial infarction, ischemic heart disease, unstable angina, or mortality in patients from real world clinical practice with newly-diagnosed type 2 diabetes and early glycemic control.

OBJECTIVES: The objective of this study was to identify factors associated with stroke, myocardial infarction (MI), all-cause mortality, or a diagnosis of ischemic heart disease (IHD) or unstable angina (UA), among patients newly-diagnosed with type 2 diabetes (T2DM) with no recent history of cardiovascular (CV) events who rapidly achieve and maintain HbA1c ≤8.0%. METHODS: Data were obtained from the Clinical Practice Research Datalink (CPRD) from January 1990 to December 2012. A nested case-control design was used with Cox proportional hazards analysis. Cases were identified by the first occurrence of stroke, MI, IHD, UA, or death within 5 years after HbA1c ≤ 8.0% was first reached (index date) following T2DM diagnosis. Controls were selected using a risk-set sampling approach and were matched 4:1 to cases using index date, exposure time, age, gender, and HbA1c at index date. RESULTS: A total of 11,426 T2DM patients met the inclusion criteria for cases. Of these, 5,261 experienced a CV event. Stroke was the most frequent CV event (40%), followed by IHD (29%), MI (22%), and UA (9%). Mean HbA1c ≥7.0% over the length of exposure (vs 6.5 to <7.0%) was associated with an increased risk of stroke, MI, and IHD. The use of anti-platelet medications at baseline was also associated with increased risk of stroke (HR = 1.82 [CI = 1.60-2.06]), MI (HR = 1.67 [CI = 1.38-2.03]), and IHD (HR = 1.85 [CI = 1.57-2.17]). Mean HbA1c < 6.0% was associated with increased risk of stroke (HR = 1.29 [CI = 1.02-1.63]) and IHD (HR = 1.65 [CI = 1.25-2.19]). Use of nitrate medications at baseline was associated with increased risk of MI (HR = 2.83 [CI = 2.24-3.57]), IHD (HR = 4.32 [CI = 3.57-5.22]), and UA (HR = 10.38 [CI = 7.67-14.03]). CONCLUSIONS: Early and sustained HbA1c control between 6.5 and <7.0% appears to be an important modifiable factor that helps reduce CV risk in patients with newly-diagnosed T2DM in real-world clinical practice.

Concordant changes of plasma and kidney microRNA in the early stages of acute kidney injury: time course in a mouse model of bilateral renal ischemia-reperfusion.

BACKGROUND: Acute kidney injury (AKI) is a syndrome characterized by the rapid loss of the kidney excretory function and is strongly associated with increased early and long-term patient morbidity and mortality. Early diagnosis of AKI is challenging; therefore we profiled plasma microRNA in an effort to identify potential diagnostic circulating markers of renal failure. The goal of the present study was to investigate the dynamic relationship of circulating and renal microRNA profiles within the first 24 hours after bilateral ischemia-reperfusion kidney injury in mice. METHODOLOGY/PRINCIPAL FINDINGS: Bilateral renal ischemia was induced in C57Bl/6 mice (n = 10 per group) by clamping the renal pedicle for 27 min. Ischemia-reperfusion caused highly reproducible, progressive, concordant elevation of miR-714, miR-1188, miR-1897-3p, miR-877*, and miR-1224 in plasma and kidneys at 3, 6 and 24 hours after acute kidney injury compared to the sham-operated mice (n = 5). These dynamics correlated with histologic findings of kidney injury and with a conventional plasma marker of renal dysfunction (creatinine). Pathway analysis revealed close association between miR-1897-3p and Nucks1 gene expression, which putative downstream targets include genes linked to renal injury, inflammation and apoptosis. CONCLUSIONS/SIGNIFICANCE: Systematic profiling of renal and plasma microRNAs in the early stages of experimental AKI provides the first step in advancing circulating microRNAs to the level of promising novel biomarkers.

Glucose metabolic trapping in mouse arteries: nonradioactive assay of atherosclerotic plaque inflammation applicable to drug discovery.

BACKGROUND: (18)F-Fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging of atherosclerosis in the clinic is based on preferential accumulation of radioactive glucose analog in atherosclerotic plaques. FDG-PET is challenging in mouse models due to limited resolution and high cost. We aimed to quantify accumulation of nonradioactive glucose metabolite, FDG-6-phosphate, in the mouse atherosclerotic plaques as a simple alternative to PET imaging. METHODOLOGY/PRINCIPAL FINDINGS: Nonradioactive FDG was injected 30 minutes before euthanasia. Arteries were dissected, and lipids were extracted. The arteries were re-extracted with 50% acetonitrile-50% methanol-0.1% formic acid. A daughter ion of FDG-6-phosphate was quantified using liquid chromatography and mass spectrometry (LC/MS/MS). Thus, both traditional (cholesterol) and novel (FDG-6-phosphate) markers were assayed in the same tissue. FDG-6-phosphate was accumulated in atherosclerotic lesions associated with carotid ligation of the Western diet fed ApoE knockout mice (5.9 times increase compare to unligated carotids, p<0.001). Treatment with the liver X receptor agonist T0901317 significantly (2.1 times, p<0.01) reduced FDG-6-phosphate accumulation 2 weeks after surgery. Anti-atherosclerotic effects were independently confirmed by reduction in lesion size, macrophage number, cholesterol ester accumulation, and macrophage proteolytic activity. CONCLUSIONS/SIGNIFICANCE: Mass spectrometry of FDG-6-phosphate in experimental atherosclerosis is consistent with plaque inflammation and provides potential translational link to the clinical studies utilizing FDG-PET imaging.

Protein kinase C inhibition ameliorates functional endothelial insulin resistance and vascular smooth muscle cell hypersensitivity to insulin in diabetic hypertensive rats.

OBJECTIVE: Insulin resistance, diabetes, and hypertension are considered elements of metabolic syndrome which is associated with vascular dysfunction. We investigated whether inhibition of protein kinase C (PKC) would affect vascular function in diabetic hypertensive (DH) rats. METHODS: A combination of type 2 diabetes and arterial hypertension was produced in male Sprague Dawley rats by intrauterine protein deprivation (IUPD) followed by high salt diet. At the age of 32 weeks, DH rats were treated for 2 weeks with the angiotensin-converting enzyme inhibitor captopril (Capto, 30 mg/kg), PKC inhibitor ruboxistaurin (RBX, 50 mg/kg) or vehicle (n = 8 per group) and blood pressure was monitored using telemetry. At the end of experiments, femoral arteries were dissected, and vascular reactivity was evaluated with isovolumic myography. RESULTS: The IUPD followed by high salt diet resulted in significant elevation of plasma glucose, plasma insulin, and blood pressure. Endothelium-dependent vascular relaxation in response to acetylcholine was blunted while vascular contraction in response to phenylephrine was enhanced in the DH rats. Neither Capto nor RBX restored endothelium-dependent vascular relaxation while both suppressed vascular contraction. Ex-vivo incubation of femoral arteries from control rats with insulin induced dose-response vasorelaxation while insulin failed to induce vasorelaxation in the DH rat arteries. In the control arteries treated with endothelial nitric oxide synthase inhibitor L-NAME, insulin induced vasoconstriction that was exacerbated in DH rats. Capto and RBX partially inhibited insulin-stimulated vascular contraction. CONCLUSION: These findings suggest that PKC inhibition ameliorates functional endothelial insulin resistance and smooth muscle cell hypersensitivity to insulin, but does not restore acetylcholine-activated endothelium-dependent vasodilation in DH rats.

Modulation of lipoprotein metabolism by inhibition of sphingomyelin synthesis in ApoE knockout mice.

Plasma sphingomyelin (SM) has been suggested as a risk factor for coronary heart disease independent of cholesterol levels. A decrease of SM in lipoproteins is known to improve the activities of lecithin:cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) in vitro. Inhibition of SM biosynthesis may reduce lipoprotein SM content and thus improve cholesterol distribution in lipoproteins by enhancing reverse cholesterol transport and clearance of triglyceride-rich lipoproteins. To examine this hypothesis, ApoE KO mice were fed a western diet and treated for 4 weeks with various concentrations of myriocin, a specific inhibitor of serine palmitoyltransferase. Myriocin treatment lowered plasma cholesterol and TG levels in a dose-dependent manner. In addition, myriocin treatment reduced cholesterol contents in VLDL and LDL and elevated HDL-cholesterol. Observed lipid-lowering effects of myriocin were associated with suppression of HMG CoA reductase and fatty acid synthase via reduced levels of SREBP-1 RNA and protein. Induction of apoAI and lecithin:cholesterol acytransferase (LCAT) in the liver by myriocin was associated with an increased HDL. Lesion area and macrophage area were also diminished in the cuffed femoral artery of ApoE KO mice. In conclusion, inhibition of sphingolipid biosynthesis can be a novel therapeutic target for dyslipidemia and atherosclerosis.

Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein E-knockout mice.

BACKGROUND: In clinical studies, sphingomyelin (SM) plasma levels correlated with the occurrence of coronary heart disease independently of plasma cholesterol levels. We hypothesized that inhibition of SM synthesis would have antiatherogenic effects. To test this hypothesis, apolipoprotein E (apoE)-knockout (KO) mice were treated with myriocin, a potent inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in SM biosynthesis. METHODS AND RESULTS: Diet-admix treatment of apoE-KO mice with myriocin in Western diet for 12 weeks lowered SM and sphinganine plasma levels. Decreases in sphinganine and SM concentrations were also observed in the liver and aorta of myriocin-treated animals compared with controls. Inhibition of de novo sphingolipid biosynthesis reduced total cholesterol and triglyceride plasma levels. Cholesterol distribution in lipoproteins demonstrated a decrease in beta-VLDL and LDL cholesterol and an increase in HDL cholesterol. Oil red O staining of total aortas demonstrated reduction of atherosclerotic lesion coverage in the myriocin-treated group. Atherosclerotic plaque area was also reduced in the aortic root and brachiocephalic artery. CONCLUSIONS: Inhibition of de novo SM biosynthesis in apoE-KO mice lowers plasma cholesterol and triglyceride levels, raises HDL cholesterol, and prevents development of atherosclerotic lesions.

Determination of hydroxyproline in plasma and tissue using electrospray mass spectrometry.

A simple and highly specific method that was developed for the determination of hydroxyproline in biological samples is described. This method could potentially be used for monitoring pathological conditions related to collagen degradation, as well as for screening remedial pharmaceuticals for efficacy. Tissue or plasma samples were prepared by hydrolysis and their hydroxyproline content was determined using spiked calibration curves and LC/MS/MS. Specificity of the method was evaluated using an API Time-Of-Flight (TOF) LC/MS to expose potential interferences. The method proposed here appears to be selective, convenient, precise (<10% R.S.D.), accurate (<10% RE), and sensitive over a linear range of 0.010-10 microg/ml.

From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I.

Atherosclerotic cardiovascular disease results in >19 million deaths annually, and coronary heart disease accounts for the majority of this toll. Despite major advances in treatment of coronary heart disease patients, a large number of victims of the disease who are apparently healthy die suddenly without prior symptoms. Available screening and diagnostic methods are insufficient to identify the victims before the event occurs. The recognition of the role of the vulnerable plaque has opened new avenues of opportunity in the field of cardiovascular medicine. This consensus document concludes the following. (1) Rupture-prone plaques are not the only vulnerable plaques. All types of atherosclerotic plaques with high likelihood of thrombotic complications and rapid progression should be considered as vulnerable plaques. We propose a classification for clinical as well as pathological evaluation of vulnerable plaques. (2) Vulnerable plaques are not the only culprit factors for the development of acute coronary syndromes, myocardial infarction, and sudden cardiac death. Vulnerable blood (prone to thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) play an important role in the outcome. Therefore, the term "vulnerable patient" may be more appropriate and is proposed now for the identification of subjects with high likelihood of developing cardiac events in the near future. (3) A quantitative method for cumulative risk assessment of vulnerable patients needs to be developed that may include variables based on plaque, blood, and myocardial vulnerability. In Part I of this consensus document, we cover the new definition of vulnerable plaque and its relationship with vulnerable patients. Part II of this consensus document focuses on vulnerable blood and vulnerable myocardium and provide an outline of overall risk assessment of vulnerable patients. Parts I and II are meant to provide a general consensus and overviews the new field of vulnerable patient. Recently developed assays (eg, C-reactive protein), imaging techniques (eg, CT and MRI), noninvasive electrophysiological tests (for vulnerable myocardium), and emerging catheters (to localize and characterize vulnerable plaque) in combination with future genomic and proteomic techniques will guide us in the search for vulnerable patients. It will also lead to the development and deployment of new therapies and ultimately to reduce the incidence of acute coronary syndromes and sudden cardiac death. We encourage healthcare policy makers to promote translational research for screening and treatment of vulnerable patients.

From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II.

Atherosclerotic cardiovascular disease results in >19 million deaths annually, and coronary heart disease accounts for the majority of this toll. Despite major advances in treatment of coronary heart disease patients, a large number of victims of the disease who are apparently healthy die suddenly without prior symptoms. Available screening and diagnostic methods are insufficient to identify the victims before the event occurs. The recognition of the role of the vulnerable plaque has opened new avenues of opportunity in the field of cardiovascular medicine. This consensus document concludes the following. (1) Rupture-prone plaques are not the only vulnerable plaques. All types of atherosclerotic plaques with high likelihood of thrombotic complications and rapid progression should be considered as vulnerable plaques. We propose a classification for clinical as well as pathological evaluation of vulnerable plaques. (2) Vulnerable plaques are not the only culprit factors for the development of acute coronary syndromes, myocardial infarction, and sudden cardiac death. Vulnerable blood (prone to thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) play an important role in the outcome. Therefore, the term "vulnerable patient" may be more appropriate and is proposed now for the identification of subjects with high likelihood of developing cardiac events in the near future. (3) A quantitative method for cumulative risk assessment of vulnerable patients needs to be developed that may include variables based on plaque, blood, and myocardial vulnerability. In Part I of this consensus document, we cover the new definition of vulnerable plaque and its relationship with vulnerable patients. Part II of this consensus document will focus on vulnerable blood and vulnerable myocardium and provide an outline of overall risk assessment of vulnerable patients. Parts I and II are meant to provide a general consensus and overviews the new field of vulnerable patient. Recently developed assays (eg, C-reactive protein), imaging techniques (eg, CT and MRI), noninvasive electrophysiological tests (for vulnerable myocardium), and emerging catheters (to localize and characterize vulnerable plaque) in combination with future genomic and proteomic techniques will guide us in the search for vulnerable patients. It will also lead to the development and deployment of new therapies and ultimately to reduce the incidence of acute coronary syndromes and sudden cardiac death. We encourage healthcare policy makers to promote translational research for screening and treatment of vulnerable patients.

How to evaluate plaque vulnerability in animal models of atherosclerosis?

Prevention of heart attack and stroke depends on detection of vulnerable plaques and development of plaque-stabilizing therapies. In turn, progress in diagnostics and treatment is contingent on our understanding of molecular mechanisms of plaque vulnerability. Animal models are essential for testing mechanistic hypotheses in a controlled manner. Currently, there is no single, golden standard animal model of a vulnerable plaque. However, the whole range of experimental approaches is readily available. It includes traditional models of atherosclerosis combined with new 'vulnerability endpoints', as well as several models featuring spontaneous or induced plaque rupture/thrombosis. This review summarizes current literature on the animal models of vulnerable atherosclerotic plaques.