The role of extracellular vesicles in neointima formation post vascular injury

Pathological neointimal growth can develop in patients as a result of vascular injury following percutaneous coronary intervention and coronary artery bypass grafting using autologous saphenous vein, leading to arterial or vein graft occlusion. Neointima formation driven by intimal hyperplasia occurs as a result of a complex interplay between molecular and cellular processes involving different cell types including endothelial cells, vascular smooth muscle cells and various inflammatory cells. Therefore, understanding the intercellular communication mechanisms underlying this process remains of fundamental importance in order to develop therapeutic strategies to preserve endothelial integrity and vascular health post coronary interventions. Extracellular vesicles (EVs), including microvesicles and exosomes, are membrane-bound particles secreted by cells which mediate intercellular signalling in physiological and pathophysiological states, however their role in neointima formation is not fully understood. The purification and characterization techniques currently used in the field are associated with many limitations which significantly hinder the ability to comprehensively study the role of specific EV types and make direct functional comparisons between EV subpopulations. In this review, the current knowledge focusing on EV signalling in neointima formation post vascular injury is discussed

AD51B in Familial Breast Cancer

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients and subsequently genotyped the mutation in additional breast cancer cases (n = 5259) and population controls (n = 3586) from Finland and Belarus. No significant association with breast cancer risk was seen in the meta-analysis of the Finnish datasets or in the large BCAC dataset. The association with previously identified risk variants rs999737, rs2588809, and rs1314913 was replicated among all breast cancer cases and also among familial cases in the BCAC dataset. The most significant association was observed for the haplotype carrying the risk-alleles of all the three SNPs both among all cases (odds ratio (OR): 1.15, 95% confidence interval (CI): 1.11–1.19, P = 8.88 x 10−16) and among familial cases (OR: 1.24, 95% CI: 1.16–1.32, P = 6.19 x 10−11), compared to the haplotype with the respective protective alleles. Our results suggest that loss-of-function mutations in RAD51B are rare, but common variation at the RAD51B region is significantly associated with familial breast cancer risk

Interactions of adenovirus vectors with blood: implications for intravascular gene therapy applications

Despite various obstacles the promise of gene therapy has begun to be realized, as demonstrated by the successful phenotypic correction of X-linked SCID in infants. Although ex vivo gene therapy is advantageous, many diseases, for example, disseminated cancers, require intravascular administration of the gene therapy vector in vivo. In this scenario, the development of sophisticated vectors suitable for targeted intravascular gene delivery is required to both improve efficacy and minimize toxicity. Vectors based on adenovirus (Ad) show immense promise because they are highly efficient in transducing non-dividing cells, can tolerate substantial genetic manipulation (eg, the incorporation of targeting agents), can be produced to high titer, do not integrate into the genome, and have undergone significant investigation in the clinic. However, the use of Ad-based vectors is limited by the inherent hepatic tropism of intravascularly administered Ad, which precludes targeted delivery to alternative organs or disease sites, and by the associated host inflammatory responses to the vector. An improved knowledge of the complex series of interactions is of fundamental importance to the field. This review discusses the current understanding of Ad vector and host interactions, as well as suitable technologies for optimizing delivery to target cells in vivo

Changes of intra-mitochondrial Ca2+ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2+ indicator targeted to mitochondria

In this study a Ca2+sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca2+-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Methods: Mitycam expression pattern and Ca2+sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca2+transient. Cytoplasmic (Fura-2) and mitochondrial Ca2+(Mitycam) fluorescence were measured simultaneously under a range of cellular Ca2+loads. Results: After 48hrs post adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca2+sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a Kd of ~220nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarisations. Time-to-peak of the mitochondrial Ca2+transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca2+release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by ~65% and increased the time to 50% decay, whilst cytosolic Ca2+transients were unchanged. The mitochondrial Ca2+uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca2+. Conclusion: The mitochondrial-targeted Ca2+probe indicates sustained and transient phases of mitochondrial Ca2+signal, which are dependent on cytoplasmic Ca2+levels and required a functional mC

Angiotensin-(1-7) and angiotensin-(1-9) inhibit vascular smooth muscle cell growth and migration in vitro and vascular remodelling in vivo

Background: Vascular smooth muscle cell(VSMC) proliferation and migration underlies the pathogenesis of atherosclerosis, vein graft failure and in-stent restenosis. Angiotensin II(AngII), acting via the AT1R, is integral in these processes. AngII is inhibited by the counter-regulatory axis of the renin angiotensin system, which is centered around the actions of angiotensin-converting-enzyme-2 and the production of Ang-(1-7) and Ang-(1-9), which act via the Mas receptor and AT2R, respectively. Here we investigated the role of Ang-(1-9) and Ang-(1-7) in primary human VSMC migration and proliferation, and in a mouse model of vascular injury. Methods: Migration was assessed via scratch assay and proliferation using the MTS-assay. Vascular injury was induced in vivo via wire injury to the left carotid artery. Ang-(1-7) and Ang-(1-9) were delivered via osmotic minipump and vascular remodeling quantified at 28 days post-injury. Results: VSMC migration and proliferation was inhibited by Ang-(1-9) and Ang-(1-7); these effects were selectively blocked by the pharmacological antagonists PD123,319 and A779, respectively, suggesting Ang-(1-9) acts via the AT2R and Ang-(1-7) via Mas. In vivo wire injury of the mouse carotid artery induced significant neointimal formation(NI) at 28 days post-injury; this was attenuated by Ang-(1-7) and Ang-(1-9) via Mas and the AT2R, respectively. Conclusion: These data demonstrate that Ang-(1-7) and Ang-(1-9) inhibit VSMC proliferation and migration in vitro and neointimal formation in vivo via the AT2R and Mas receptor, respectively. Here we demonstrate for the first time a direct biological effect of Ang-(1-9) within the vasculature. These findings highlight the potential of Ang-(1-9) and Ang-(1-7) as therapeutic agents in vascular injury

Prevention of coronary in-stent restenosis and vein graft failure: Does vascular gene therapy have a role?

Coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI), including stent insertion, are established therapies in both acute coronary syndromes (ACS) and symptomatic chronic coronary artery disease refractory to pharmacological therapy. These continually advancing treatments remain limited by failure of conduit grafts in CABG and by restenosis or thrombosis of stented vessel segments in PCI caused by neointimal hyperplasia, impaired endothelialisation and accelerated atherosclerosis. While pharmacological and technological advancements have improved patient outcomes following both procedures, when grafts or stents fail these result in significant health burdens. In this review we discuss the pathophysiology of vein graft disease and in-stent restenosis, gene therapy vector development and design, and translation from pre-clinical animal models through human clinical trials. We identify the key issues that are currently preventing vascular gene therapy from interfacing with clinical use and introduce the areas of research attempting to overcome these

Angiotensin-(1-9) antagonises Angiotensin II-induced cardiac remodeling via the angiotensin type 2 receptor

The renin-angiotensin system (RAS) regulates cardiovascular physiology mainly via angiotensin II (AngII) engaging the angiotensin type 1 or type 2 receptors (AT1R and AT2R). Classical AngII actions are AT1R-mediated, while the AT2R may counteract AT1R signalling. However recent identification of the ACE2/Ang-(1-7)/Mas has led to the definition of a counter-regulatory axis of the RAS. Novel peptides of the counter-regulatory axis of the RAS, angiotensin 1-7 [Ang-(1-7)] and angiotensin 1-9 [Ang-(1-9)], have been identified as potential therapeutic molecules. Ang-(1-7) has been shown to antagonise the pathological actions of AngII through the receptor Mas. Recently, we showed that Ang-(1-9) has an anti-hypertrophic effect on AngII-induced rat neonatal cardiomyocytes, signalling through the angiotensin type 2 receptor (AT2R)1. Furthermore, infusion of Ang-(1-9) via osmotic minipumps to the stroke-prone spontaneously hypertensive rat reduced cardiac fibrosis via the AT2R)2. To further investigate the actions of Ang-(1-9) in an acute model of hypertension we infused AngII (24µg/kg/hr) in mice to induce hypertension. Ang-(1-9) (48µg/kg/hr), Ang-(1-9)+PD123,319 (AT2R blocker) (20mg/kg/day), or water (control) were co-infused with AngII for 2 weeks via osmotic minipumps. Blood pressure was monitored via radiotelemetry (for radiotelemetry probe and osmotic minipump implantation mice were anesthetized with 1.5% isofluorane in 1 L/min O2 for the duration of the surgical implantation procedure). Significant increases in mean arterial pressure (MAP) were observed in AngII infused mice compared to control (control 108.8 ± 5.7 mmHg; AngII 125.1 ± 8.4 mmHg; p><0.05; n= 6 mice; two way ANOVA) however co-infusion of Ang-(1-9) or Ang-(1-9) and PD123,319 did not affect AngII-induced hypertension (AngII + Ang-(1-9) 122.4 ± 10.3 mmHg; AngII + Ang-(1-9) + PD123,319 118.3 ± 6.8mmHg; n=6 mice; two way ANOVA). However, Ang-(1-9) co-infusion was able to reduce AngII-induced cardiac hypertrophy (ratio heart weight/tibia length: Control 9.43 ± 1.2 mg; AngII 11.69 ± 0.6 mg; Ang-(1-9) 8.22 ± 0.7 mg; p<0.01; n=6 mice; one way ANOVA). Staining of cardiac sections with wheat germ agglutinin and measurement of cardiomyocyte size confirmed these results (Control 25.73 ± 3.8 µm; AngII 28.78 ± 3.8 µm; Ang-(1-9) 24.6 ± 4.8 µm; p<0.05; one way ANOVA). Cardiac fibrosis was assessed by picrosirius red staining and quantified by image analysis software. Mice co-infused with Ang-(1-9) showed a reduction in cardiac fibrosis when compared to AngII-infused animals (control 0.12 ± 0.16%; AngII 1.28 ± 2.2%; Ang-(1-9) 0.52 ± 0.75%; p<0.05; one way ANOVA). The effects of Ang-(1-9) were reversed by PD123,319 co-infusion. In summary Ang-(1-9) was able to reduce cardiac hypertrophy and fibrosis in an acute animal model of hypertension without effecting blood pressure. These data support a direct biological role for Ang-(1-9) in cardiac remodeling and highlights Ang-(1-9) as a potential new therapeutic target in cardiovascular disease

Angiotensin-(1-9) antagonises Angiotensin II-induced cardiac remodeling via the angiotensin type 2 receptor

The renin-angiotensin system (RAS) regulates cardiovascular physiology mainly via angiotensin II (AngII) engaging the angiotensin type 1 or type 2 receptors (AT1R and AT2R). Classical AngII actions are AT1R-mediated, while the AT2R may counteract AT1R signalling. However recent identification of the ACE2/Ang-(1-7)/Mas has led to the definition of a counter-regulatory axis of the RAS. Novel peptides of the counter-regulatory axis of the RAS, angiotensin 1-7 [Ang-(1-7)] and angiotensin 1-9 [Ang-(1-9)], have been identified as potential therapeutic molecules. Ang-(1-7) has been shown to antagonise the pathological actions of AngII through the receptor Mas. Recently, we showed that Ang-(1-9) has an anti-hypertrophic effect on AngII-induced rat neonatal cardiomyocytes, signalling through the angiotensin type 2 receptor (AT2R)1. Furthermore, infusion of Ang-(1-9) via osmotic minipumps to the stroke-prone spontaneously hypertensive rat reduced cardiac fibrosis via the AT2R)2. To further investigate the actions of Ang-(1-9) in an acute model of hypertension we infused AngII (24µg/kg/hr) in mice to induce hypertension. Ang-(1-9) (48µg/kg/hr), Ang-(1-9)+PD123,319 (AT2R blocker) (20mg/kg/day), or water (control) were co-infused with AngII for 2 weeks via osmotic minipumps. Blood pressure was monitored via radiotelemetry (for radiotelemetry probe and osmotic minipump implantation mice were anesthetized with 1.5% isofluorane in 1 L/min O2 for the duration of the surgical implantation procedure). Significant increases in mean arterial pressure (MAP) were observed in AngII infused mice compared to control (control 108.8 ± 5.7 mmHg; AngII 125.1 ± 8.4 mmHg; p><0.05; n= 6 mice; two way ANOVA) however co-infusion of Ang-(1-9) or Ang-(1-9) and PD123,319 did not affect AngII-induced hypertension (AngII + Ang-(1-9) 122.4 ± 10.3 mmHg; AngII + Ang-(1-9) + PD123,319 118.3 ± 6.8mmHg; n=6 mice; two way ANOVA). However, Ang-(1-9) co-infusion was able to reduce AngII-induced cardiac hypertrophy (ratio heart weight/tibia length: Control 9.43 ± 1.2 mg; AngII 11.69 ± 0.6 mg; Ang-(1-9) 8.22 ± 0.7 mg; p<0.01; n=6 mice; one way ANOVA). Staining of cardiac sections with wheat germ agglutinin and measurement of cardiomyocyte size confirmed these results (Control 25.73 ± 3.8 µm; AngII 28.78 ± 3.8 µm; Ang-(1-9) 24.6 ± 4.8 µm; p<0.05; one way ANOVA). Cardiac fibrosis was assessed by picrosirius red staining and quantified by image analysis software. Mice co-infused with Ang-(1-9) showed a reduction in cardiac fibrosis when compared to AngII-infused animals (control 0.12 ± 0.16%; AngII 1.28 ± 2.2%; Ang-(1-9) 0.52 ± 0.75%; p<0.05; one way ANOVA). The effects of Ang-(1-9) were reversed by PD123,319 co-infusion. In summary Ang-(1-9) was able to reduce cardiac hypertrophy and fibrosis in an acute animal model of hypertension without effecting blood pressure. These data support a direct biological role for Ang-(1-9) in cardiac remodeling and highlights Ang-(1-9) as a potential new therapeutic target in cardiovascular disease

Angiotensin-(1-9) attenuates cardiac fibrosis in the stroke-prone spontaneously hypertensive rat via the angiotensin type 2 receptor

The renin-angiotensin system regulates cardiovascular physiology via angiotensin II engaging the angiotensin type 1 or type 2 receptors. Classic actions are type 1 receptor mediated, whereas the type 2 receptor may counteract type 1 receptor activity. Angiotensin-converting enzyme 2 metabolizes angiotensin II to angiotensin-(1-7) and angiotensin I to angiotensin-(1-9). Angiotensin-(1-7) antagonizes angiotensin II actions via the receptor Mas. Angiotensin-(1-9) was shown recently to block cardiomyocyte hypertrophy via the angiotensin type 2 receptor. Here, we investigated in vivo effects of angiotensin-(1-9) via the angiotensin type 2 receptor. Angiotensin-(1-9) (100 ng/kg per minute) with or without the angiotensin type 2 receptor antagonist PD123 319 (100 ng/kg per minute) or PD123 319 alone was infused via osmotic minipump for 4 weeks into stroke-prone spontaneously hypertensive rats. We measured blood pressure by radiotelemetry and cardiac structure and function by echocardiography. Angiotensin-(1-9) did not affect blood pressure or left ventricular mass index but reduced cardiac fibrosis by 50% (P<0.01) through modulating collagen I expression, reversed by PD123 319 coinfusion. In addition, angiotensin-(1-9) inhibited fibroblast proliferation in vitro in a PD123 319-sensitive manner. Aortic myography revealed that angiotensin-(1-9) significantly increased contraction to phenylephrine compared with controls after N-nitro-l-arginine methyl ester treatment, an effect abolished by PD123 319 coinfusion (area under the curve: angiotensin-(1-9) N-nitro-l-arginine methyl ester=98.9±11.8%; control+N-nitro-l-arginine methyl ester=74.0±10.4%; P<0.01), suggesting that angiotensin-(1-9) improved basal NO bioavailability in an angiotensin type 2 receptor–sensitive manner. In summary, angiotensin-(1-9) reduced cardiac fibrosis and altered aortic contraction via the angiotensin type 2 receptor supporting a direct role for angiotensin-(1-9) in the renin-angiotensin system