Chiara Giannarelli

Real-time Measurement System for Evaluation of the Carotid Intima-Media Thickness With a Robust Edge Operator

Objective. The purpose of this report is to describe an automatic real‐time system for evaluation of the carotid intima‐media thickness (CIMT) characterized by 3 main features: minimal interobserver and intraobserver variability, real‐time capabilities, and great robustness against noise. Methods. One hundred fifty carotid B‐mode ultrasound images were used to validate the system. Two skilled operators were involved in the analysis. Agreement with the gold standard, defined as the mean of 2 manual measurements of a skilled operator, and the interobserver and intraobserver variability were quantitatively evaluated by regression analysis and Bland‐Altman statistics. Results. The automatic measure of the CIMT showed a mean bias ± SD of 0.001 ± 0.035 mm toward the manual measurement. The intraobserver variability, evaluated with Bland‐Altman plots, showed a bias that was not significantly different from 0, whereas the SD of the differences was greater in the manual analysis (0.038 mm) than in the automatic analysis (0.006 mm). For interobserver variability, the automatic measurement had a bias that was not significantly different from 0, with a satisfactory SD of the differences (0.01 mm), whereas in the manual measurement, a little bias was present (0.012 mm), and the SD of the differences was noticeably greater (0.044 mm). Conclusions. The CIMT has been accepted as a noninvasive marker of early vascular alteration. At present, the manual approach is largely used to estimate CIMT values. However, that method is highly operator dependent and time‐consuming. For these reasons, we developed a new system for the CIMT measurement that conjugates precision with real‐time analysis, thus providing considerable advantages in clinical practice.

Non-Invasive Diagnostic Tools for Investigating Endothelial Dysfunction

The endothelium is not merely a barrier but it plays a key role in the maintenance of vascular homeostasis. A dysfunctional endothelium is an early marker of the development of atherosclerotic changes and can also contribute to cardiovascular events. Vascular reactivity tests represent the most widely used methods in the clinical assessment of endothelial function and in the last two decades, several methodologies were developed to study it non invasively in the peripheral macrocirculation (conduit arteries) and microcirculation (resistance arteries and arterioles). This review will centre on the most relevant available non-invasive techniques in the research on endothelial function, their advantages and limitations. Flow mediated dilation (FMD) of the brachial artery by ultrasounds is the most widely used vascular test to assess endothelium-dependent vasodilation. Other approaches include measurement of microcirculatory reactive hyperaemia by forearm venous pletysmography or digital pulse amplitude tonometry, response to beta2 agonist by applanation tonometry or digital photoplethysmography and several test by skin laser doppler. It appears that FMD is the most reproducible test when an appropriate and accurate methodology is applied. Recently, post-ischemic vasodilation in the cavernous arteries was also suggested to study endothelial function in patients with erectile dysfunction. Systemic markers proposed as measures of NO biology, inflammatory cytokines, adhesion molecules, or markers of endothelial damage and repair have only a very limited role as a result of biological and assay availability and variability, these factors currently have a limited role in the assessment of individual patients. The optimal methodology for investigating the multifaceted aspects of endothelial dysfunction is still under debate. Therefore, no available test to assess endothelial function has sufficient sensitivity and specificity to be used yet in clinical practice. Only the growing concordant results from different reproducible and reliable non-invasive methods exploring endothelial function with different stimuli will support and strengthen experimental findings, thus providing conclusive answers in this area of research.

Obesity in the childhood: a link to adult hypertension.

The rapid increasing prevalence of obesity worldwide represents a serious health hazard. Obesity predisposes to increased risk for diabetes, hypertension, renal failure. Direct mechanisms link visceral adiposity and the atherosclerosis process through the action of adipose-derived proinflammatory cytokines. In particular, hypertension can be considered the most important cardiovascular risk factor linking obesity to the development of cardiovascular disease. Obesity among children and adolescents has also reaching epidemic proportions in the industrialized world. Childhood obesity strongly predisposes to cardiovascular adult mortality. Recent reports documented a tracking of blood pressure from childhood to adulthood and obesity occurring in young age plays a crucial pathogenic role. Indeed, fighting overweight and obesity in the pediatric and adolescent age may prevent the occurrence of adults with hypertension and cardiovascular disease. The main strategies for prevention and treatment of overweight and obesity in childhood, which need to involve community, school and family, are the promotion of lifestyle interventions, including as a correct dietary approach, rich in fruit and vegetables and low-fat dairy products, and physical activity.

Systems Pharmacology of Adverse Event Mitigation by Drug Combinations

Drug combinations that mitigate adverse events were identified using the FDA Adverse Event Reporting System, and one combination, exenatide and rosiglitazone, was tested in a rodent model. Two Drugs: Better Than One? Everyone has seen the commercial, where a drug is advertised as the much-awaited treatment for a disease. At the end of the commercial, there is a long list of adverse events (or side effects) that may affect anything from your heart to your eyesight. Surprisingly, the addition of a second drug can mitigate the side effects of the first drug, such that the combination is actually safer for the patient. To search for those mitigating combinations, Zhao and colleagues devised a computational method for scanning the Food and Drug Administration’s Adverse Event Reporting System (FAERS). This database is freely available and contains millions of records of drug-induced adverse events reported by patients, doctors, hospitals, lawyers, and drug companies. Thus, it represents a potentially useful source of beneficial drug combinations. The authors focused on rosiglitazone—a drug that effectively controls blood glucose levels in diabetic patients, but has been associated with myocardial infarction (MI). By searching through FAERS data, Zhao et al. found that when rosiglitazone was prescribed in combination with exenatide—another drug for treating type 2 diabetes—there were significantly fewer reports of MI as an adverse event. A cell biological interaction network was then developed to look for mechanisms by which rosiglitazone + exenatide affected the heart. The rosiglitazone target PPARγ was plugged into this network, and plasminogen activator inhibitor-1 (PAI-1) was obtained as a potential point of convergence between the two drugs. To test this hypothesis, the authors administered drugs to a mouse model of type 2 diabetes. Mice treated with rosiglitazone alone showed a marked increase in PAI-1 levels, whereas mice treated with the drug combination had significantly decreased levels of PAI-1, similar to those found in untreated mice. FAERS is self-reported and thus may contain some inaccurate data. Nevertheless, it could be a useful tool for generating meaningful hypotheses from human data and for then testing in vivo in clinically relevant disease models, as shown by Zhao et al. Animal models can provide information about drug mechanism of action, and prospective clinical trials will confirm that such combinations can indeed be translated to people to prevent adverse events. Drugs are designed for therapy, but medication-related adverse events are common, and risk/benefit analysis is critical for determining clinical use. Rosiglitazone, an efficacious antidiabetic drug, is associated with increased myocardial infarctions (MIs), thus limiting its usage. Because diabetic patients are often prescribed multiple drugs, we searched for usage of a second drug (“drug B”) in the Food and Drug Administration’s Adverse Event Reporting System (FAERS) that could mitigate the risk of rosiglitazone (“drug A”)–associated MI. In FAERS, rosiglitazone usage is associated with increased occurrence of MI, but its combination with exenatide significantly reduces rosiglitazone-associated MI. Clinical data from the Mount Sinai Data Warehouse support the observations from FAERS. Analysis for confounding factors using logistic regression showed that they were not responsible for the observed effect. Using cell biological networks, we predicted that the mitigating effect of exenatide on rosiglitazone-associated MI could occur through clotting regulation. Data we obtained from the db/db mouse model agreed with the network prediction. To determine whether polypharmacology could generally be a basis for adverse event mitigation, we analyzed the FAERS database for other drug combinations wherein drug B reduced serious adverse events reported with drug A usage such as anaphylactic shock and suicidality. This analysis revealed 19,133 combinations that could be further studied. We conclude that this type of crowdsourced approach of using databases like FAERS can help to identify drugs that could potentially be repurposed for mitigation of serious adverse events.

Recombinant HDLMilano exerts greater anti-inflammatory and plaque stabilizing properties than HDLwild-type

OBJECTIVE The aim of this study was to compare the effects of HDL(Milano) and HDL(wild-type), on regression and stabilization of atherosclerosis. METHODS Atherosclerotic New Zealand White rabbits received 2 infusions, 4 days apart, of HDL(Milano) (75mg/kg of apoA-I(Milano)), HDL(wild-type) (75mg/kg apoA-I(wild-type)) or placebo. Pre- and post-treatment plaque volume was assessed by MRI. Markers of plaque vulnerability and inflammation were evaluated. Liver and aortic cholesterol content, aortic ABCA-1 and liver SR-BI were quantified. The effect of apoA-I Milano and wild-type proteins on MCP-1 and COX-2 expression by macrophages was evaluated in vitro. RESULTS Both forms of HDL induced aortic plaque regression (-4.1% and -2.6% vs. pre-treatment in HDL(Milano) and HDL(wild-type) respectively, p<0.001 and p=0.009). A similar reduction in cholesterol content of aorta and liver was observed with both treatments vs. placebo. The expression of aortic ABCA-1 and hepatic SR-BI was significantly higher in both treated groups vs. placebo. A significantly reduced plaque macrophage density was observed in the HDL(Milano) vs. both HDL(wild-type) and placebo groups. Plaque levels of COX-2, MCP-1, Caspase-3 antigen and MMP-2 activity were significantly reduced in the HDL(Milano) vs. both HDL(wild-type) and placebo groups. In vitro studies showed that apoA-I(Milano) protein significantly reduced expression of COX-2 and MCP-1 in oxLDL loaded macrophages vs. apoA-I(wild-type). CONCLUSIONS Despite a similar effect on acute plaque regression, the infusion of HDL(Milano) exerts superior anti-inflammatory and plaque stabilizing effects than HDL(wild-type) in the short term.

Atorvastatin Prevents Endothelial Dysfunction in Mesenteric Arteries From Spontaneously Hypertensive Rats: Role of Cyclooxygenase 2-Derived Contracting Prostanoids

We investigated the effect of atorvastatin on cyclooxygenase (COX) contribution to endothelial dysfunction in spontaneously hypertensive rat (SHR) mesenteric resistance arteries. Atorvastatin (10 mg/kg per day, oral gavage) or its vehicle was administered for 2 weeks to male SHR or Wistar-Kyoto rats. Endothelial function of mesenteric arteries was assessed by pressurized myograph. In Wistar-Kyoto rats, relaxation to acetylcholine was inhibited by NG-nitro-l-arginine methyl ester and unaffected by SC-560 (COX-1 inhibitor), DuP-697 (COX-2 inhibitor), or ascorbic acid. In SHRs, the response to acetylcholine was attenuated, less sensitive to NG-nitro-l-arginine methyl ester, unaffected by SC-560, and enhanced by DuP-697 or SQ-29548 (thromboxane-prostanoid receptor antagonist) to a similar extent. Endothelium-dependent relaxation was normalized by ascorbic acid or apocynin (NADPH oxidase inhibitor), which also restored the inhibition by NG-nitro-l-arginine methyl ester. In atorvastatin-treated SHRs, relaxation to acetylcholine was normalized, fully sensitive to NG-nitro-l-arginine methyl ester, and not affected by SC-560, DuP-697, SQ 29548, or antioxidants. Dihydroethidium assay showed an increased intravascular superoxide generation in SHRs, which was abrogated by atorvastatin. RT-PCR revealed a COX-2 induction in SHR arteries, which was downregulated by atorvastatin. The release of prostacyclin and 8-isoprostane was higher from SHR than Wistar-Kyoto mesenteric vessels. COX-2 inhibition and apocynin decreased 8-isoprostane without affecting prostacyclin levels. Atorvastatin increased phosphorylated extracellular signal–regulated kinase 1/2, pAkt, peNOS1177, and inducible NO synthase levels in SHR mesenteric vessels and decreased 8-isoprostane release. In conclusion, COX-2–derived 8-isoprostane contributes to endothelial dysfunction in SHR mesenteric arteries. Atorvastatin restores NO availability by increasing phosphorylated extracellular signal–regulated kinase 1/2, pAkt, peNOS1177, and inducible NO synthase levels and by abrogating vascular NADPH oxidase–driven superoxide production, which also results in a downregulation of COX-2–dependent 8-isoprostane generation.

Ramipril dose-dependently increases nitric oxide availability in the radial artery of essential hypertension patients.

Design and participants A double-blind, crossover, randomized study was designed to evaluate the effect of 3-month treatment with a lower versus a higher antihypertensive dosage of ramipril (5 or 10 mg/day) on nitric oxide (NO)-dependent vasodilation in 46 untreated patients with essential hypertension. Radial artery flow-mediated dilation (FMD), before and after the intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA), to block NO synthase, and the response to sublingual glyceril trinitrate (GTN, 25 μg) were measured at baseline and after the two treatment periods as a change in artery diameter (computerized system from ultrasound scans). Plasma angiotensin II and oxidative stress markers were also assessed. Results FMD was significantly (P < 0.01) lower in hypertensive patients (4.6 ± 1.8%) than in normotensive subjects (7.1 ± 2.6%), whereas the response to GTN was similar. L-NMMA significantly (P < 0.001) inhibited FMD in normotensive but not in hypertensive subjects. Mean 24-h ambulatory blood pressure, plasma angiotensin II and oxidative stress marker levels were similarly reduced at the end of the two treatment periods. Both dosages of ramipril significantly (P < 0.001) increased FMD (5 mg: 5.9 ± 2.1%; 10 mg: 6.3 ± 2.4%) without modifying the response to GTN. However, compared with baseline (11 ± 19%), the inhibiting effect of L-NMMA on FMD (NO-dependent FMD) was significantly (P < 0.01) greater with ramipril 10 mg (49 ± 12%) than 5 mg per day (38 ± 15%). The improvement in FMD and NO-dependent FMD was not related to changes in plasma levels of angiotensin II or markers of oxidative stress. Conclusion Treatment with ramipril at a higher dosage induced a greater improvement in NO-dependent vasodilation compared with the lower antihypertensive dosage in hypertensive patients.

Cyclooxygenase-1 Is Involved in Endothelial Dysfunction of Mesenteric Small Arteries From Angiotensin II–Infused Mice

Angiotensin II induces endothelial dysfunction by reducing NO availability and increasing reactive oxygen species. We assessed whether cyclooxygenase (COX)-1 or COX-2 participate in the angiotensin II-induced endothelial dysfunction in murine mesenteric small arteries and examined the role of reduced nicotinamide-adenine dinucleotide phosphate-dependent reactive oxygen species production. Mice received angiotensin II (600 ng/kg per minute, SC), saline (controls), angiotensin II + apocynin (reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, 2.5 mg/day), or apocynin alone for 2 weeks. Endothelial function of mesenteric arteries was assessed by pressurized myograph. In controls, acetylcholine-induced relaxation was inhibited by NG-monomethyl-l-arginine and unaffected by DFU (COX-2 inhibitor), SC-560 (COX-1 inhibitor), or ascorbic acid. In angiotensin II-infused animals, the attenuated response to acetylcholine was less sensitive to NG-monomethyl-l-arginine, unaffected by DFU, and enhanced by SC-560 and, similarly, by SQ-29548, a thromboxane-prostanoid receptor antagonist. Moreover, response to acetylcholine was unchanged by ozagrel, a thromboxane synthase inhibitor, and normalized by ascorbic acid. Apocynin prevented the angiotensin II-induced vascular dysfunctions. In angiotensin II-infused mice, RT-PCR analysis showed a significant COX-2 downregulation, whereas COX-1 expression was upregulated. These changes were unaffected by apocynin. Modulation of COX isoform by angiotensin II was also documented by immunohistochemistry. In small mesenteric vessels, the reduced NO availability and oxidant excess, which characterize endothelial dysfunction secondary to angiotensin II, are associated with a reduced COX-2 and an increased COX-1 function and expression. Angiotensin II causes an oxidative stress-independent COX-1 overexpression, whereas angiotensin II-mediated oxidant excess production stimulates COX-1 activity to produce a contracting prostanoid endowed with agonist activity on thromboxane-prostanoid receptors.

Endothelial dysfunction and vascular disease in later life

The endothelium plays a primary role in the modulation of vascular tone and structure, through production of the relaxing factor nitric oxide, which acts by protecting the vessel wall from the development of atherosclerosis and thrombosis. A dysfunctioning endothelium, characterized by reduced nitric oxide availability, can be a promoter of atherosclerosis. Ageing is a well-documented cardiovascular risk factor. One of the possible physiopathological mechanisms whereby increasing age may lead to cardiovascular damage is the promotion of endothelial dysfunction. In humans, age-related impairment in endothelium-dependent vasodilation has been well documented in different vascular districts and involves the impairment of nitric oxide activity secondary to oxidative stress generation. Regular physical training is an important non-pharmacological intervention which protects the vascular endothelium from ageing-related alterations and ameliorates the cardiovascular risk profile among the elderly population.

Cardiometabolic risk loci share downstream cis- and trans-gene regulation across tissues and diseases

Genetic variation and coronary artery disease Most genetic variants lie outside protein-coding genes, but their effects, especially in human health, are not well understood. Franzén et al. examined gene expression in tissues affected by coronary artery disease (CAD). They found that individuals with loci that have been associated with CAD in genome-wide analyses had different patterns of tissue-specific gene expression than individuals without these genetic variants. Similarly, tissues not associated with CAD did not have CAD-like expression patterns. Thus, tissue-specific data can be used to dissect the genetic effects that predispose individuals to CAD. Science, this issue p. 827 A gene expression survey in patients with coronary artery disease reveals how genetic variation affects the risk of heart failure. Genome-wide association studies (GWAS) have identified hundreds of cardiometabolic disease (CMD) risk loci. However, they contribute little to genetic variance, and most downstream gene-regulatory mechanisms are unknown. We genotyped and RNA-sequenced vascular and metabolic tissues from 600 coronary artery disease patients in the Stockholm-Tartu Atherosclerosis Reverse Networks Engineering Task study (STARNET). Gene expression traits associated with CMD risk single-nucleotide polymorphism (SNPs) identified by GWAS were more extensively found in STARNET than in tissue- and disease-unspecific gene-tissue expression studies, indicating sharing of downstream cis-/trans-gene regulation across tissues and CMDs. In contrast, the regulatory effects of other GWAS risk SNPs were tissue-specific; abdominal fat emerged as an important gene-regulatory site for blood lipids, such as for the low-density lipoprotein cholesterol and coronary artery disease risk gene PCSK9. STARNET provides insights into gene-regulatory mechanisms for CMD risk loci, facilitating their translation into opportunities for diagnosis, therapy, and prevention.