Naked plasmid DNA (pDNA) and short interfering RNA (siRNA) duplexes were transduced into adult murine heart by means of sonoporation using the third-generation microbubble, BR14. Plasmid DNAs carrying luciferase, b-galactosidase (b-gal), or enhanced green fluorescent protein (EGFP) reporter genes were mixed with BR14 and injected percutaneously into the left ventricular (LV) cavity of C57BL/6 mice while exposed to transthoracic ultrasound at 1 MHz for 60 s. Sonoporation at an output intensity of 2.0 W/cm 2 and a 50% pulse duty ratio resulted in the highest luciferase expression in the heart. Histological examinations revealed significant expression of the b-gal and EGFP reporters in the subendocardial myocardium of LV. Intraventricular co-injection of siRNA-GFP and BR14 with concomitant ultrasonic exposure resulted in substantial reduction in EGFP expression in the coronary artery in EGFP transgenic mice. The present method may be applicable to gain-of-function and loss-of-function genetic engineering in vivo of adult murine heart.

BackgroundDespite the importance of the renin-angiotensin (Ang) system in abdominal aortic aneurysm (AAA) pathogenesis, strategies targeting this system to prevent clinical aneurysm progression remain controversial and unproven. We compared the relative efficacy of two Ang II type 1 receptor blockers, telmisartan and irbesartan, in limiting experimental AAAs in distinct mouse models of aneurysm disease.Methodology/Principal FindingsAAAs were induced using either 1) Ang II subcutaneous infusion (1000 ng/kg/min) for 28 days in male ApoE−/− mice, or 2) transient intra-aortic porcine pancreatic elastase infusion in male C57BL/6 mice. One week prior to AAA creation, mice started to daily receive irbesartan (50 mg/kg), telmisartan (10 mg/kg), fluvastatin (40 mg/kg), bosentan (100 mg/kg), doxycycline (100 mg/kg) or vehicle alone. Efficacy was determined via serial in vivo aortic diameter measurements, histopathology and gene expression analysis at sacrifice. Aortic aneurysms developed in 67% of Ang II-infused ApoE−/− mice fed with standard chow and water alone (n = 15), and 40% died of rupture. Strikingly, no telmisartan-treated mouse developed an AAA (n = 14). Both telmisartan and irbesartan limited aneurysm enlargement, medial elastolysis, smooth muscle attenuation, macrophage infiltration, adventitial neocapillary formation, and the expression of proteinases and proinflammatory mediators. Doxycycline, fluvastatin and bosentan did not influence aneurysm progression. Telmisartan was also highly effective in intra-aortic porcine pancreatic elastase infusion-induced AAAs, a second AAA model that did not require exogenous Ang II infusion.Conclusion/SignificanceTelmisartan suppresses experimental aneurysms in a model-independent manner and may prove valuable in limiting clinical disease progression.

Objective Macrophages are critical contributors in abdominal aortic aneurysm (AAA) disease. We examined the ability of MKEY, a peptide inhibitor of CXCL4-CCL5 interaction, to influence AAA progression in murine models. Methods and Results AAAs were created in 10-week-old male C57BL/6 mice by transient infrarenal aortic porcine pancreatic elastase (PPE) infusion. Mice were treated with MKEY via intravenous injection either 1) before PPE infusion, or 2) after aneurysm initiation. Immunostaining demonstrated CCL5 and CCR5 expression on aneurysmal aortae and mural monocytes/macrophages, respectively. MKEY treatment partially inhibited transmural AAA migration of adaptively transferred leukocytes in recipient mice. While all vehicle-pretreated mice developed AAA, aneurysms formed in only 60% (3/5) and 14% (1/7) of mice pretreated with MKEY at 10 and 20 mg /kg, respectively. MKEY pretreatment reduced aortic diameter enlargement, preserved medial elastin fibers and smooth muscle cells, and attenuated mural macrophage infiltration, angiogenesis, and aortic MMP2 & 9 expression following PPE infusion. MKEY initiated after PPE infusion also stabilized and/or reduced enlargement of existing AAAs. Finally, MKEY treatment was effective in limiting AAA formation following angiotensin II infusion in apolipoprotein E deficient mice. Conclusion MKEY suppresses AAA formation and progression in two complementary experimental models. Peptide inhibition of CXCL4-CCL5 interactions may represent a viable translational strategy to limit progression of human AAA disease.

Purpose Inflammation and angiogenesis are important contributors to vascular disease. We evaluated imaging both of these biological processes, using Arg–Gly–Asp (RGD)-conjugated human ferritin nanoparticles (HFn), in experimental carotid and abdominal aortic aneurysm (AAA) disease. Procedures Macrophage-rich carotid lesions were induced by ligation in hyperlipidemic and diabetic FVB mice (n=16). AAAs were induced by angiotensin II infusion in apoE−/− mice (n=10). HFn, with or without RGD peptide, was labeled with Cy5.5 and injected intravenously for near-infrared fluorescence imaging. Results RGD-HFn showed significantly higher signal than HFn in diseased carotids and AAAs relative to non-diseased regions, both in situ (carotid: 1.88±0.30 vs. 1.17±0.10, p=0.04; AAA: 2.59±0.24 vs. 1.82±0.16, p=0.03) and ex vivo. Histology showed RGD-HFn colocalized with macrophages in carotids and both macrophages and neoangiogenesis in AAA lesions. Conclusions RGD-HFn enhances vascular molecular imaging by targeting both vascular inflammation and angiogenesis, and allows more comprehensive detection of high-risk atherosclerotic and aneurysmal vascular diseases.

Objective: We examined the pathogenic significance of VEGF (vascular endothelial growth factor)-A in experimental abdominal aortic aneurysms (AAAs) and the translational value of pharmacological VEGF-A or its receptor inhibition in aneurysm suppression. Approaches and Results: AAAs were created in male C57BL/6J mice via intra-aortic elastase infusion. Soluble VEGFR (VEGF receptor)-2 extracellular ligand-binding domain (delivered in Ad [adenovirus]-VEGFR-2), anti–VEGF-A mAb (monoclonal antibody), and sunitinib were used to sequester VEGF-A, neutralize VEGF-A, and inhibit receptor tyrosine kinase activity, respectively. Influences on AAAs were assessed using ultrasonography and histopathology. In vitro transwell migration and quantitative reverse transcription polymerase chain reaction assays were used to assess myeloid cell chemotaxis and mRNA expression, respectively. Abundant VEGF-A mRNA and VEGF-A–positive cells were present in aneurysmal aortae. Sequestration of VEGF-A by Ad-VEGFR-2 prevented AAA formation, with attenuation of medial elastolysis and smooth muscle depletion, mural angiogenesis and monocyte/macrophage infiltration. Treatment with anti–VEGF-A mAb prevented AAA formation without affecting further progression of established AAAs. Sunitinib therapy substantially mitigated both AAA formation and further progression of established AAAs, attenuated aneurysmal aortic MMP2 (matrix metalloproteinase) and MMP9 protein expression, inhibited inflammatory monocyte and neutrophil chemotaxis to VEGF-A, and reduced MMP2, MMP9, and VEGF-A mRNA expression in macrophages and smooth muscle cells in vitro. Additionally, sunitinib treatment reduced circulating monocytes in aneurysmal mice. Conclusions: VEGF-A and its receptors contribute to experimental AAA formation by suppressing mural angiogenesis, MMP and VEGF-A production, myeloid cell chemotaxis, and circulating monocytes. Pharmacological inhibition of receptor tyrosine kinases by sunitinib or related compounds may provide novel opportunities for clinical aneurysm suppression.

PurposeTo evaluate Arg‐Gly‐Asp (RGD)‐conjugated human ferritin (HFn) iron oxide nanoparticles for in vivo magnetic resonance imaging (MRI) of vascular inflammation and angiogenesis in experimental carotid disease and abdominal aortic aneurysm (AAA).Materials and MethodsHFn was genetically engineered to express the RGD peptide and Fe3O4 nanoparticles were chemically synthesized inside the engineered HFn (RGD‐HFn). Macrophage‐rich left carotid lesions were induced by ligation in FVB mice made hyperlipidemic and diabetic (n = 14), with the contralateral right carotid serving as control. Murine AAAs were created by continuous angiotensin II infusion in ApoE‐deficient mice (n = 12), while control mice underwent saline infusion (n = 8). All mice were imaged before and after intravenous injection with either RGD‐HFn‐Fe3O4 or HFn‐Fe3O4 using a gradient‐echo sequence on a whole‐body 3T clinical scanner, followed by histological analysis. The nanoparticle accumulation was assessed by the extent of T2*‐induced carotid lumen reduction (% lumen loss) or aortic T2*‐weighted signal intensity reduction (% SI [signal intensity] loss).ResultsRGD‐HFn‐Fe3O4 was taken up more than HFn‐Fe3O4 in both the ligated left carotid arteries (% lumen loss; 69 ± 9% vs. 36 ± 7%, P = 0.01) and AAAs (% SI loss; 47 ± 6% vs. 20 ± 5%, P = 0.01). The AAA % SI loss correlated positively with AAA size (r = 0.89, P < 0.001). Histology confirmed the greater accumulation and colocalization of RGD‐HFn‐Fe3O4 to both vascular macrophages and endothelial cells.ConclusionRGD‐HFn‐Fe3O4 enhances in vivo MRI by targeting both vascular inflammation and angiogenesis, and provides a promising translatable MRI approach to detect high‐risk atherosclerotic and aneurysmal vascular diseases. Level of Evidence: 1J. Magn. Reson. Imaging 2017;45:1144–1153

Operative outcomes showed no significant differences between the groups except for early recovery after TEVAR. Long-term survival was similar between groups; however, TEVAR had inferior reintervention free rate.

AVNeo is suitable for patients with AS considering its early and mid-term outcomes. Verification of long-term outcomes and reliability is necessary.