Rafaela F. da Silva

Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE-/- mice

AIMSnWall shear stress differentially regulates the arginase pathway in carotid arteries perfused ex vivo. Specific patterns of wall shear stress can locally determine atherosclerotic plaque size and composition in vivo. The present work investigates the effects of arginase inhibition on shear stress induced plaque composition.nnnMETHODS AND RESULTSnCarotid arteries of apolipoprotein E deficient mice were exposed to high (HSS), low (LSS) and oscillatory (OSS) shear stress conditions by the placement of a local shear stress modifier device for 9 weeks with or without the administration of the arginase inhibitor N-ω-Hydroxy-nor-L-arginine (nor-Noha) (10 mg/kg, i.p., 5 days/week). Carotid arginase activity was measured by colorimetric determination of urea. Atherosclerotic plaque size and composition, arginase expression and cellular localization were assessed by immunohistochemistry. Arginase activity was significantly increased in both LSS and OSS regions as compared to HSS. In the lesions, arginase II isoform co-localized preferentially with EC. Inhibition of arginase by nor-Noha decreased arginase activity and reduced plaque size in both LSS and OSS regions. Arginase inhibition affected mainly the composition of plaques developed in LSS regions by decreasing the total vascular ROS, the number of macrophages, apoptosis rate, lipid and collagen contents.nnnCONCLUSIONSnArginase activity is modulated by patterns of wall shear stress in vivo. Chronic inhibition of vascular arginase decreased the size of atherosclerotic lesions in both OSS and LSS regions, whereas changes on plaque composition were more pronounced in plaques induced by LSS. We identified wall shear stress as a key biomechanical regulator of arginase during plaque formation and stability.

Mas receptor deficiency is associated with worsening of lipid profile and severe hepatic steatosis in ApoE knockout mice

The classical renin-angiotensin system pathway has been recently updated with the identification of additional molecules [such as angiotensin converting enzyme 2, ANG-(1-7), and Mas receptor] that might improve some pathophysiological processes in chronic inflammatory diseases. In the present study, we focused on the potential protective role of Mas receptor activation on mouse lipid profile, liver steatosis, and atherogenesis. Mas/apolipoprotein E (ApoE)-double-knockout (DKO) mice (based on C57BL/6 strain of 20 wk of age) were fed under normal diet and compared with aged-matched Mas and ApoE-single-knockout (KO), as well as wild-type mice. Mas/ApoE double deficiency was associated with increased serum levels of atherogenic fractions of cholesterol, triglycerides, and fasting glucose compared with wild-type or single KO. Serum levels of HDL or leptin in DKO were lower than in other groups. Hepatic lipid content as well as alanine aminotransferase serum levels were increased in DKO compared with wild-type or single-KO animals. Accordingly, the hepatic protein content of mediators related to atherosclerotic inflammation, such as peroxisome proliferator-activated receptor-α and liver X receptor, was altered in an adverse way in DKO compared with ApoE-KO. On the other hand, DKO mice did not display increased atherogenesis and intraplaque inflammation compared with ApoE-KO group. In conclusion, Mas deletion in ApoE-KO mice was associated with development of severe liver steatosis and dyslipidemia without affecting concomitant atherosclerosis. Mas receptor activation might represent promising strategies for future treatments targeting both hepatic and metabolic alterations in chronic conditions clustering these disorders.

Conversion of human M-CSF macrophages into foam cells reduces their proinflammatory responses to classical M1-polarizing activation

BACKGROUNDnIn atherosclerotic lesions, cholesterol-laden macrophage foam cells are formed and exposed to M1- and M2-polarizing factors. However, the effects of the polarizing factors on the proinflammatory and the anti-inflammatory potential of foam cells are not known.nnnOBJECTIVEnTo investigate the effects of M1- and M2-polarizing factors on the expression of pro- and anti-inflammatory genes in cultured human macrophage foam cells.nnnMETHODS AND RESULTSnHuman monocytes were differentiated into macrophages in the presence of M-CSF, and then converted into cholesterol-loaded foam cells by incubation with acetylated LDL. The generated macrophages and foam cells were polarized into the M1 phenotype by classical activation with LPS and IFN-γ, or into the M2 phenotype by alternative activation with IL-4. When subjected to the M1-polarizing factors, the macrophages responded by typical upregulation of several key proinflammatory genes (TNFA, IL1B, CXCL8, CCL19, and COX2), while the anti-inflammatory genes (MRC1, CCL17, and IL10) displayed variable responses. The foam cells, again, showed a weaker response to the M1-polarizing factors, as indicated by reduced upregulation of the proinflammatory genes, reduced secretion of TNF-α, and a trend towards lower NF-κB activity. When subjected to alternative M2 polarization, both macrophages and foam cells responded by a typical upregulation of the anti-inflammatory genes, which was of equal magnitude in both cell types.nnnCONCLUSIONSnConversion of cultured human macrophages into foam cells suppresses their proinflammatory responses to M1-polarizing factors. Thus, in M1-polarizing microenvironments of atherosclerotic lesions, foam cell formation may locally weaken the macrophage-dependent inflammatory component of atherogenesis.

Diminazene protects corpus cavernosum against hypercholesterolemia-induced injury.

INTRODUCTIONnAngiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin angiotensin system, which breaks down angiotensin II and forms angiotensin-(1-7). In erectile tissues, it has been documented that angiotensin II contributes to the development of erectile dysfunction (ED), while treatment with angiotensin-(1-7) improves penile erection. However, the expression and function of ACE2 in erectile tissues have never been investigated.nnnAIMnHere, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia-induced corpus cavernosum (CC) injury.nnnMETHODSnHypercholesterolemic apolipoprotein E knockout (ApoE(-/-) ) mice, a well-known model of ED, were treated with diminazene aceturate (DIZE), an ACE2 activator compound, or vehicle for 3 weeks. Reactive oxygen species (ROS), collagen content, and protein expression of ACE2, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) subunits were evaluated in the penis of DIZE-treated and untreated ApoE(-/-) mice. Functional studies were performed in CC strips.nnnMAIN OUTCOME MEASURESnACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures.nnnRESULTSnACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, ACE2 was downregulated in penis of hypercholesterolemic mice with ED, suggesting a protective role of ACE2 on the CC homeostasis. In accordance with that, pharmacological ACE2 activation by DIZE treatment reduced ROS production and NADPH oxidase expression, and elevated nNOS and eNOS expression and NO bioavailability in the penis of ApoE(-/-) mice. Additionally, DIZE decreased collagen content within the CC. These beneficial actions of DIZE on the CC were not accompanied by improvements in atherosclerotic plaque size or serum lipid profile.nnnCONCLUSIONnACE2 is expressed in erectile tissue and its reduction is associated with hypercholesterolemia-induced ED. Additionally, treatment with DIZE improved hypercholesterolemia-induced CC injury, suggesting ACE2 as a potential target for treating ED. .

Diminazene enhances stability of atherosclerotic plaques in ApoE-deficient mice

Angiotensin (Ang) II contributes to the development of atherosclerosis, while Ang-(1-7) has atheroprotective actions. Accordingly, angiotensin-converting enzyme 2 (ACE2), which breaks-down Ang II and forms Ang-(1-7), has been suggested as a target against atherosclerosis. Here we investigated the actions of diminazene, a recently developed ACE2 activator compound, in a model of vulnerable atherosclerotic plaque. Atherosclerotic plaque formation was induced in the carotid artery of ApoE-deficient mice by a shear stress (SS) modifier device. The animals were treated with diminazene (15mg/kg/day) or vehicle. ACE2 was strongly expressed in the aortic root and low SS-induced carotid plaques, but poorly expressed in the oscillatory SS-induced carotid plaques. Diminazene treatment did not change the lesion size, but ameliorated the composition of aortic root and low SS-induced carotid plaques by increasing collagen content and decreasing both MMP-9 expression and macrophage infiltration. Interestingly, these beneficial effects were not observed in the oscillatory SS-induced plaque. Additionally, diminazene treatment decreased intraplaque ICAM-1 and VCAM-1 expression, circulating cytokine and chemokine levels and serum triglycerides. In summary, ACE2 was distinctively expressed in atherosclerotic plaques, which depends on the local pattern of shear stress. Moreover, diminazene treatment enhances the stability of atherosclerotic plaques.

Inflammatory Cardiovascular Risk Biomarkers: Update on Novelties and Limitations

Evidence from Framingham studies showed that some disorders and conditions (such as hypertension, hyperlipidaemia, smoking, diabetes, old age, and male sex) are particularly useful to estimate the cardiovascular (CV) risk of acute ischemic events [1] and are currently considered as the “traditional” cardiovascular risk factors. This led to the development of several clinically based CV risk stratification tools, and the Framingham risk score is one of the most commonly used CV risk stratification tools nowadays [2]. However, these “traditional” cardiovascular risk factors were shown to be suboptimal for proper CV risk stratification due to low specificity and sensitivity [3–6]. Therefore, a novel concept of “global” cardiovascular vulnerability has been suggested to better predict acute cardiovascular events [7, 8]. Interestingly, this approach was particularly focused on “the clinical and laboratory complexity of the patient” instead of “a single risk factor.” Several pathophysiological parameters have been proposed as new cardiovascular risk factors potentially improving the assessment of patient vulnerability [7, 8]. Although some controversies still exist, the atherosclerotic role of inflammatory biomarkers (such as C-reactive protein (CRP), cytokines, and chemokines) [9–12] has been shown within atherosclerotic plaques, in the systemic circulation or in the peripheral ischemic tissues in both in vivo and in vitro models. More recently, novel inflammatory mediators (such as circulating autoantibodies and hormones) have been also identified [13, 14]. These soluble mediators have been shown to trigger several atherosclerotic functions of both inflammatory and vascular cells [15]. On the other hand, the mobilization of protective cell subsets might also counterbalance atheroprogression, thus limiting the chronic inflammatory processes and improving cardiovascular outcomes [16]. These protective aspects might be particularly relevant when atherosclerosis is associated with concomitant inflammatory conditions (such as rheumatoid arthritis, infections, and diabetes), which seem to further accelerate atherogenesis towards final acute ischemic complications or arterial aneurysms [17–19]. This special issue focused on new soluble mediators as promising candidates to better assess the cardiovascular risk. Importantly, the limitations of the potential clinical use of these systemic and intraplaque inflammatory molecules influencing atheroprogression have also been discussed. In particular, E. Lupia and coworkers revised the clinical role of thrombopoietin (a humoral growth factor activating platelets) as a promising biomarker of cardiovascular injury. P. Kunes and colleagues developed interesting findings on the controversial role of pentraxin family (which includes CRP) in the inflammatory response. The authors focused on the newly discovered pentraxin 3 and suggested paradoxical issues that will probably be validated in the near future. Drs. D. Vasic and the D. Walcher from the University of Ulm (Germany) revised the potential proatherosclerotic activity of C-peptide as a predictor of cardiovascular risk in diabetic subjects. This paper focused on a hot-topic issue in cardiovascular research. In fact, only very recently, C-peptide (previously considered as a product of cleavage of proinsulin) has been proposed as an active factor favouring atherosclerosis. Despite some limitations on the molecular mechanisms (the C-peptide receptor remains to be identified), this molecule might activate different leukocyte subsets in atherogenesis. Among these cells, the different types of macrophages appear as a relevant target for inflammatory mediators. T. Gui and coworkers discussed this issue and provided an interesting and comprehensive review on the impact of macrophages in both early and advanced phases of atherogenesis. The authors also suggested these cells as promising biomarkers of plaque vulnerability. Another review article of the present issue mainly focused on the proteolytic mechanisms regulating intraplaque remodelling potentially favouring the formation of abdominal aortic aneurysms. Drs. Z.- Z. Li and Q.-Y. Dai focused on direct activities mediated by nicotine via its receptor (nicotinic acetylcholine receptor) on plaque inflammation, angiogenesis, and smooth muscle cell dysfunction. The paper of Z. Qu and colleagues further developed the proinflammatory reactions underlying abdominal aortic aneurysm formation in advanced atherosclerosis. Some receptors of sphingosine-1-phosphate (S1P, a recently discovered lysophospholipid) were shown to play a crucial role in human abdominal aorta aneurysms as compared with normal aorta control tissues. In particular, S1P3 receptor was significantly upregulated in human abdominal aortic aneurysms, while S1P2 receptor was downregulated as compared to normal aortic samples. Although the molecular mechanisms remain unexplored (parallel expression of other inflammatory mediators was not investigated), this observational study identified a potential novel cardiovascular biomarker (S1P) in advanced atherosclerosis. Differently from this paper, K.-Karatolios and coworkers focused their study on well-known cytokines and growth factors. Surprisingly, the authors showed that the levels of vascular endothelial growth factor (VEGF) and human basic fibroblast growth factor (bFGF) in pericardial effusions of patients with autoreactive or viral inflammation were significantly higher as compared to patients with coronary artery disease (CAD). No significant difference was shown for inflammatory cytokines. Although the underlying molecular mechanisms remain to be investigated, these two growth factors might be more promising biomarkers of pericardial inflammation than “traditional” cytokines. After a diffuse discussion on soluble mediators potentially increasing the cardiovascular risk, R. Wyderka and coworkers focused their investigation on the mobilization of protective CD34+CXC4+ stem/progenitor cells in humans after an acute myocardial infarction. The authors clearly showed that this process was positively correlated with the improvements of values of the left ventricular ejection fraction at 1-year of followup, suggesting a beneficial activity of these cells in myocardial repair. n nIn both inflammatory micro- and macroenvironments characterizing atherosclerosis, some tissues might also produce some unexpected molecules potentially contrasting with physiological paradigms. M. L. Sirico and coworkers showed that human adipocytes can express and synthesize albumin. This paper was selected to highlight the potentialities of adipose tissue as an inflammatory organ capable of ectopically producing a large variety of mediators during atherogenesis. The present issue includes by the paper of C. Falcone and colleagues investigating the potential activity as biomarkers of soluble Receptor for Advanced Glycation End products (sRAGE) in patients with hypertension and increased cardiovascular risk. The authors showed that antihypertensive treatments might affect sRAGE plasma levels. All the papers included in the present issue focused on both novelty and limitations of promising inflammatory biomarkers that in the near future might be used in the clinical practice to improve cardiovascular disease prevention. We hope that the reader will find some useful inputs for developing research and updating knowledge on cardiovascular pathophysiology.

Neuronal nitric oxide synthase contributes to the normalization of blood pressure in medicated hypertensive patients

Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and besides NO, generates H2O2. nNOS has been proposed to contribute to the control of blood pressure in healthy humans. The aim of this study was to verify the hypothesis that nNOS can contribute to the control of vascular relaxation and blood pressure in hypertensive patients undergoing drug treatment. The study was conducted in resistance mesenteric arteries from 63 individuals, as follows: 1) normotensive patients; 2) controlled hypertensive patients (patients on antihypertensive treatment with blood pressure normalized); 3) uncontrolled hypertensive patients (patients on antihypertensive treatment that remained hypertensive). Only mesenteric arteries from uncontrolled hypertensive patients showed impaired endothelium-dependent vasorelaxation in response to acetylcholine (ACh). Selective nNOS blockade with inhibitor 1 and catalase, which decomposes H2O2, decreased vasorelaxation in the three groups. However, the inhibitory effect was greater in controlled hypertensive patients. Decreased eNOS expression was detected in both uncontrolled and controlled hypertensive groups. Interestingly nNOS expression and ACh-stimulated H2O2 production were greater in controlled hypertensive patients, than in the other groups. ACh-stimulated NO production was lower in controlled hypertensive when compared to normotensive patients, while uncontrolled hypertensive patients showed the lowest levels. Catalase and nNOS blockade inhibited ACh-induced H2O2 production. In conclusion, nNOS-derived H2O2 contributes to the endothelium-dependent vascular relaxation in human resistance mesenteric arteries. The endothelial dysfunction observed in uncontrolled hypertensive patients involves decreased eNOS expression and NO production. The normalization of vascular relaxation and blood pressure in controlled hypertensive patients involves increased nNOS-derived H2O2 and NO production.

Age-related changes in vascular responses to angiotensin-(1-7) in female mice

The vasodilatory effect of angiotensin-(1-7) seems to vary between sexes, and estradiol (E2) can modulate the magnitude of the Ang-(1-7) vasodilatory response in female rats. However, there are few studies addressing the influence of sex on the age-related vasodilatory effect of Ang-(1-7). Here, we evaluated the vasodilatory response to Ang-(1-7) on vascular ageing. Ang-(1-7) dose–response curves were determined in mice aortic rings from males (old and young) and females (E2 treated/non-treated old and young) mounted in an isolated organ chamber. Abdominal aortic rings were used for protein expression analysis and determination of reactive oxygen species (ROS) and nitric oxide (NO) production. Our results showed that the Ang-(1-7) vasodilatory effect was absent in aorta from old females, contrasting with a full response in vessels from young females. The Ang-(1-7) vasodilatory effect was restored by E2 replacement in old females. A robust increase in Mas receptor, SOD2, NRF-2 and NOX2 expression was observed in aorta from old females, which was normalized by E2. This effect of E2 was also associated with lower production of ROS and normal levels of NO. In conclusion, our data demonstrated that pathways involved in the Ang-(1-7) vasodilatory response in female mice is affected by hormonal changes in ageing and rescued by E2.

Lack of interferon-gamma attenuates foreign body reaction to subcutaneous implants in mice: Lack of interferon-gamma attenuates

Subcutaneous implantation of synthetic materials and biomedical devices often induces abnormal tissue healing - the foreign body reaction-which impairs their function. In particular, Interferon-γ (IFN-γ) is a critical endogenous mediator of inflammation and plays a key role in a wide variety of biological responses including tissue healing. However, the contribution of endogenous IFN-γ on different features of the foreign body response induced by synthetic implants regarding neovascularization, inflammation, and fibrogenesis is not well known. Here, we evaluated inflammatory angiogenesis and fibrogenesis induced by implantation of polyether-polyurethane sponges in mice targeted disrupted of the interferon-γ gene (IFN-γ-/- ) and wild-type (WT). The hemoglobin content, the number of vessels, and blood flow (evaluated by LDPI-laser Doppler perfusion imaging) were decreased in the implants from IFN-γ-/- as compared to WT mice. Likewise, neutrophils and macrophages accumulation (MPO and NAG activities, respectively) was decreased in IFN-γ-/- implants. Interestingly, while the local content of VEGF, TNF-α, CXCL-1/KC, as measured by ELISA, and iNOS expression, as measured by qPCR, were significantly reduced, the content of IL-10 was greatly increased in the implants from IFN-γ-/- mice as compared to WT mice. No alterations were observed in CCL-2/MCP-1 levels. Lastly, the collagen deposition, assessed by Picro-Sirius red-stained histological sections, was also reduced in IFN-γ-/- implants. Altogether, these data suggest that IFN-γ activity contributes to inflammatory angiogenesis and fibrogenesis in synthetic implants and that lack of IFN-γ expression attenuates foreign body reaction to implants in mice.

Apelin-13 treatment enhances the stability of atherosclerotic plaques

Apelin is an endogenous peptidergic system which modulates cardiovascular function. Recent studies pointed out a fundamental contribution of apelin on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define a beneficial or deleterious role. To better understand apelin function on atherosclerosis, we aimed to investigate apelin‐13 treatment effects on atherosclerotic plaques composition.