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Aortic aneurysm is a silent but life-threatening disease, whose pathogenesis remains poorly understood. Aneurysm models have been induced in small animals to study its pathogenesis, Johnston WF et al. successfully induced a novel model of thoracic aortic aneurysms (TAA) by periadventitial application of elastase in mice. We comment on this model according to our experiment. We hypothesize that endogenous MMPs, especially MMP2, play a vital role in complex repair process of aneurysmal wall, which should be a key target in the investigation and treatment of aortic aneurysms., Highlights • Aortic aneurysm model formed mainly owing to the matrix destruction by exogenous elastase rather than endogenous MMP. • MMPs may play a positive role in remodeling of aneurysmal wall. • MMP2 should be a key target for investigation and treatment of aortic aneurysm.

We are quite interested in the important review by M. Parvizi and M.C. Harmsen, published on February 23, 2015, in Stem Cells and Development [1]. The authors pointed out that studies failed to explain the initial trigger and initiation of abdominal aortic aneurysm (AAA) in animal studies. The typical chicken or egg question also arouses our enormous interest. To answer this question, we successfully induce a novel model of enlarging AAA in rabbits by a combination of elastase incubation and aortic coarctation [2]. Our model successfully overcomes the self-healing phenomenon in elastase-induced AAA and indicated that hemodynamic change is vital to the initiation and progression of AAA by a certain mechanism. The onset of AAA diseases involves the activation of the endothelial cells [1]. A decreased endothelial recovery was shown in our experiment; perhaps, endothelial cells are vital to the initiation of AAA under the influence of hemodynamics. The authors pointed out that smooth muscle cells (SMCs), the main workhorse of an artery, not only serve a contractile role but in addition produce, deposit, and continuously remodel the vascular extracellular matrix. We quite appreciate this point and hypothesize that SMCs account for self-healing of AAA model. In our previous studies, matrix metalloproteinase (MMP)2 kept moderate expression during a 5-month follow-up [3], and a similar expression was shown in enlarging AAA; however, the increased expressions are not proportionate to the enlargement of AAA. Aneurysm develops after infusion of elastase due to an inflammatory cascade that ultimately leads to matrix degradation by MMPs. This classical theory indicates that MMPs play an evil role in AAA formation, and it seems scientific to treat AAA by inhibiting MMPs. However, we hypothesize that MMPs, especially MMP2, play a positive role in the complex remodeling of aneurysmal wall, which then cause a self-healing of AAA. Shen et al. found that MMP2 plays two opposing roles in aortic wall remodeling [4]. Interestingly, so many treatments, including the MMP inhibitor, achieved perfect results in animal studies; however, few are of benefit to human AAA diseases. It seems more reasonable to treat AAA by enhancing the repair progress of aneurysmal wall, such as promoting SMCs adjacent to aneurysm migration and proliferation to some rational degree. Besides, the ‘‘response to injury’’ hypothesis suggests that SMCs migrate and subsequently proliferate after injury, and the contractile SMCs be modulated back into the synthetic phenotype and participate in tissue repair [5]. It seems feasible and beneficial to promote and restore the phenotype of the affected medial SMCs.

To the editor,We are quite interested in the important article by Mata and col-leagues, published on November 5, 2014, in Cardiovascular Pathology [1].Their research indicates that pretreatment with doxycycline via gavage(30 mg/kg per day) inhibited the activity of matrix metalloproteinases(MMP) and the inflammatory response, and may play an important rolein the prevention of the development of abdominal aortic aneurysm(AAA). Of note, this conclusion was drawn in a novel enlarging AAAmodelinrats,notinatraditionalrat modelinducedbyelastaseperfusion.We drew inspiration from the authors' research and hypothesizedthat hemodynamic change caused by coarctation is vital to the progres-sionofAAA.NovelrabbitAAAmode lwasinducedandenlargedprogres-sively after a combination of low concentration elastase incubation andaortic coarctation [2], which successfully overcomes the self-healingphenomenon reported by Origuchi and colleagues [3].Itisexcitingbutalso confusing for us that the authors achieved perfect results in thischallenging rat AAA model. Interestingly, the authors showed a typicalAAA with a dramatic large diameter in Fig. 1A and B at 3 dayspostsurgery, and the diameter did not increase significantly from Day 3to Day 15. In our rabbit AAA model, a dilatation ratio of more than311% was seldom found even by Week 16, and the diameter enlargedgradually during follow-up. Can the authors please comment on this?Aneurysmdevelopsafterinfusionofelastaseduetoaninflammatorycascade that ultimately results in matrix degradation by MMPs. ThisAAA model indicates that MMPs play an “evil” role in AAA formation,anditisscientifictopreventAAAbyinhibitingMMPs.Theenlargementoftheaortacoincideswithinflammatorycellinfiltrationafewdaysafterinfusionin thismodel[4]. We speculate that aneurysmismainly duetothe expansion of the weakened wall when exposed to pulsatile bloodflow, and inflammatory cell infiltration is the result of damage to theaorta by chemical corrosion rather than cause of AAA progression.MMP9-positive staining and macrophages in filtration were shown inthe elastase-incubated aorta by Week 2. However, both expressionsdecreased dramatically thereafter [5]. MMP2 expression increased afterelastase exposure and kept moderate expression during a 5-monthfollow-up [5]. These similar expressions were also seen in our enlargingAAA model; however, the increased expressions are not proportionate tothe enlargement of AAA. It seems that hemodynamic change plays a vitalrole in the progression of AAA rather than endogenous MMPs. MMP2plays an important role in smooth muscle cell (SMC) migration andproliferation, which may account for the self-healing seen in an elastase-induced AAA model in rabbits. Endogenous MMPs, such as MMP2 andMMP9, are important to the complex remodeling process.It is maybe confusing and contradictory to inhibit MMPs foraneurysm treatment. It seems more reasonable to treat AAA byenhancing the repair progress of aneurismal wall, such as promotingSMC migration and proliferation to some rational degree, which mayneed to up-regulate MMPs expression rather than down-regulatethem. We look forward to the authors' comments.Yonghua BiDepartment of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhou 450052, ChinaDepartment of RadiologyThe First Affiliated Hospital of China Medical UniversityShenyang 110001, ChinaKey Laboratory of Diagnostic Imaging and Interventional Radiology ofLiaoning Province, Shenyang, ChinaHongshan ZhongKe Xu

Atherosclerosis, the leading cause of most cardiovascular disease, is a progressive multifaceted inflammatory disease characterized by extracellular matrix degradation and extensive remodeling of artery wall. However, its mechanism has not been completely understood, and animal models are useful to study its pathogenetic process. An analysis of literature on the nature of atherosclerosis indicates that focal accumulation of smooth muscle cells (SMCs) into the intima by plasma factors is fundamental to the entire process of plaque growth. In our previous study, vascular SMCs proliferation was obvious in elastase-induced aorta by day 15, which led to intimal hyperplasia and regression of rabbit aneurysm. Model induced by combination of balloon injury and an atherogenic diet in rabbits is the conventional, but most largely used experimental model of atherosclerosis. Since proliferation and accumulation of intimal SMCs are found in elastase-induced aorta, and hypercholesterolemia is usually induced by cholesterol-rich diets in rabbits, a novel atherosclerosis model may be induced by combination periaortic elastase incubation and cholesterol-rich diet.

The overall prognosis for malignant glioma is extremely poor, and treatment options are limited in part because of multidrug resistant proteins. Our previous findings suggest low intensity ultrasound (LIUS) can induce apoptosis of glioma cells. Given this finding, we were interested in determining if LIUS could help treat glioma by inhibiting multidrug resistant proteins, and if so, which pathways are involved. In this study, the toxicity sensitivity and multidrug resistance proteins of glioma induced by LIUS were investigated using CCK-8, immunohistochemistry, immunofluorency, and RT-PCR in tissue samples and cultured cells. LIUS inhibited increase of C6 cells in an intensity- and time-dependent manner. The toxicity sensitivity of C6 cells increased significantly after LIUS sonication (intensity of 142.0 mW/cm(2)) or Doxorubicin (DOX) at different concentration, particularly by the combination of LIUS sonication and DOX. The expressions of P-gp and MRP1 decreased significantly post-sonication at intensity of 142.0 mW/cm(2) both in vitro and in vivo. The expressions of p110 delta (PI3K), NF-κB-p65, Akt/PKB, and p-Akt/PKB were downregulated by LIUS sonication and DOX treatment separately or in combination at the same parameters in rat glioma. These results indicate that LIUS could increase the toxicity sensitivity of glioma by down-regulating the expressions of P-gp and MRP1, which might be mediated by the PI3K/Akt/NF-κB pathway.

Background The purpose of this study was to introduce a novel, simple and effective technique for creating a reliable rabbit model of abdominal aortic aneurysm (AAA) via a combination of periaortic calcium chloride (CaCl2) and elastase incubation. Methods Forty-eight New Zealand white rabbits were divided into four groups. The AAA model was developed via a 20-minute periaortic incubation of CaCl2 (0.5 mol/L) and elastase (1 Unit/µL) in a 1.5-cm aortic segment (Group CE). A single incubation of CaCl2 (Group C) or elastase (Group E) and a sham operation group (Sham Group) were used for the controls. Diameter was measured by serial digital subtraction angiography imaging on days 5, 15 and 30. Animals were sacrificed on day 5 and day 30 for histopathological and immunohistochemical studies. Results All animals in Group CE developed aneurysm, with an average dilation ratio of 65.3% ± 8.9% on day 5, 86.5% ± 28.7% on day 15 and 203.6% ± 39.1% on day 30. No aneurysm was found in Group C, and only one aneurysm was seen on day 5 in Group E. Group CE exhibited less intima-media thickness, endothelial recovery, elastin and smooth muscle cell (SMC) content, but stronger expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and RAM11 compared to the controls. Conclusions The novel rabbit model of AAA created by using a combination of periaortic CaCl2 and elastase incubation is simple and effective to perform and is valuable for elucidating AAA mechanisms and therapeutic interventions in experimental studies.

Objectives To modify the method for creating an abdominal aortic aneurysm in rabbits, and to study its performance. Materials and methods A total of 24 New Zealand white rabbits were induced topically with 10 μl of porcine elastase (0, 0.1, 5 and 10 units μl−1) to define the optimal concentration (groups A–D). Twelve aneurysms were induced with 10 units μl−1 of 10 μl elastase to serve as a follow-up group (group E) to serve as a follow-up. A 1.5-cm aortic segment was isolated and induced with elastase solution for 30 min. Results All animals in groups D and E developed AAA by day 5. Aneurysms in Group E were stable over 100 days. Partial destruction to disappearance of elastic lamellae and smooth muscle cells (SMCs) was seen in elastase-treated animals by day 5. Regenerated elastin and proliferated SMCs were present in group E. Matrix metalloproteinases 2 and 9 and RAM11 showed strong expression in group D, but expression decreased in group E after day 15. Conclusions The rabbit AAA model induced via topical application of porcine elastase at 10 units μl−1 for 30 min appears easy and simple, with shorter induction and more rapid aortic dilation. The model is stable over 100 days and is useful to study the formation and progress of AAAs.

The objective of this study was to determine the possibility of creating a novel animal model of abdominal aortic aneurysm (AAA) in rabbits by the periarterial application of papain.Twelve New Zealand white rabbits were randomized into two groups: (1) the papain group, which received 2mg of papain (n = 8) and (2) the control group, which received physiologic saline solution (n = 4). A 1-cm aortic segment proximal to the bifurcation was isolated, and its adventitia was incubated with papain for 20 minutes. The rabbits underwent intravenous digital subtraction angiography (IVDSA) 5 and 21 days after the operation. The animals were then humanely killed for histomorphometric and immunohistochemical studies.All animals in the papain group developed AAA, with an average aneurysm diameter of 4.0 ± 0.6 and 4.1 ± 0.4 mm on days 5 and 21, respectively. No aneurysms were seen in the control group. On day 5, the papain-incubated aortas exhibited thinned and disorganized aortic walls, with decreased smooth muscle cells (SMCs) and fragmented and almost nonexistent elastic lamella. Media thickening, intimal hyperplasia, and smooth muscle cell regeneration were obvious on day 21. Immunostaining of matrix metalloproteinase (MMP)-9 and RAM11 showed strong expression in the papain group. On the contrary, the control group did not present histologic alterations and showed almost no expression of MMP-9 and RAM11.A novel in vivo rabbit model of AAA can be induced through periarterial application of papain for 20 minutes. This model is similar to an elastase-induced aneurysm model and could be useful to clarify AAA pathogenesis and endovascular treatment intervention.

Low frequency and intensity ultrasound (LFU) sonication can selectively induce brain tumor cell apoptosis without damaging neural cells, while also enhancing drug delivery to brain tumors. To explore the underlying mechanisms of related pathways in LFU-induced apoptosis, we investigated the expression of proteins associated with LFU-induced apoptosis. C6 cells were used for in vitro experiments and C6 tumor-bearing rats were used during in vivo experiments. 3-[4.5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to detect C6 cell viability in vitro. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis was used to check the apoptotic cells, and they were counted and analyzed both in vitro and in vivo. Transmission electron microscopy (TEM) was used to illustrate the ultrastructure of apoptotic nuclei of cancer cells in vivo. The expressions of caspase-3, Bcl-2, and survivin proteins were assessed by immunofluorescence, immunohistochemistry and Western blot analysis in vivo. C6 cell viability decrease was statistically significant; the numbers of apoptotic C6 cells in the LFU sonication groups were higher than those in the control group both in vitro and in vivo. The expression of caspase-3 increased, yet the expressions of Bcl-2 and survivin decreased significantly 6h after LFU sonication, compared with the control group in vivo. This study suggests that LFU can induce apoptosis in vitro and in vivo, and that three signaling proteins, caspase-3, Bcl-2, and survivin, might be involved in LFU-induced apoptosis.

PURPOSE: To present a new endovascular technique for placing handmade partially covered stent-grafts in rabbit aortas that is promising for experimental study of direct gene delivery to the aortic wall. METHODS: A 7-mm-diameter Z-stent made from the inner core of a guidewire was covered with a 7-mm-diameter, 10-mm-long polyester fabric tube (2268 mL porosity). To decrease stent profile and make delivery possible through a 6-F introducer, one third of the fabric was cut away to form a partially covered polyester stent-graft. Two stent-grafts were delivered sequentially into the descending thoracic aorta of 12 male Japanese White rabbits; a third device was positioned in each aorta so that the orifices of the celiac trunk and superior mesenteric artery were not occluded. RESULTS: The implantation was successful in 10 animals. One rabbit died during the procedure due to sheath laceration of the infrarenal abdominal aorta. Another animal died within 2 days after the procedure owing to occlusion of the celiac trunk by graft fabric. At 2 weeks, the stent-grafts in the 10 surviving rabbits remained patent, and there was no migration. Gross examination of the lumens showed that both the metal stent and the polyester graft material were completely covered with thin transparent tissue, without massive thrombosis. Histological staining revealed incomplete neointima formation between the stent-graft and the aorta. Incomplete linear endothelial cells on the luminal side of the tissue ingrowth into the stent-graft were also observed. Foreign body giant cells and macrophages represented inflammatory reactions related to the graft material. CONCLUSION: Partially covered stent-grafts can be safely placed in relatively small animals and potentially used in research.

ObjectiveStent grafts are increasingly recognized as useful devices for endovascular repair of aortic aneurysms and other vascular diseases. Stent graft-mediated gene delivery into the vascular wall is expected to improve their therapeutic effects. This study evaluated the efficacy of genetically engineered cationized gelatin (CG) hydrogel-coated partially-covered polyester stent grafts that facilitate delivery of an expression plasmid DNA in rabbit aortic wall.MethodsPartially covered polyester stent grafts coated with CG hydrogel impregnated with 10.0 mg/mL of β-galactosidase (LacZ)-expression plasmid vector (pCAGGS-LacZ) or empty vector (pCAGGS) solutions were implanted via the femoral artery in rabbit balloon-injured aortas. The aortic segments were removed at 1, 3, or 7 days (4 rabbits/each group) after implantation and evaluated for the transgene (LacZ) delivery and expression by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) staining. Partially-covered polyester stent grafts coated with CG hydrogel impregnated with various amounts (0.1 mg/mL, 1.0 mg/mL, and 10.0 mg/mL) of pCAGGS-LacZ or pCAGGS were also implanted in rabbits' balloon-injured aortas (4 rabbits/each group) to evaluate transgene delivery and expression in the aortic wall 3 days after implantation. The difference of transgene efficiency among each group was compared using one-way analysis of variance (ANOVA) and Newman-Keuls' test according to the result of quantitative RT-PCR.ResultsIn all animals, LacZ gene transduction into the aortic wall was detected at the implantation site of pCAGGS-LacZ-loaded, but not pCAGGS-loaded, stent grafts. LacZ expression was not detected in aortic segments immediately proximal or distal to the implanted pCAGGS-LacZ-loaded stent graft or remote organs including the brain, heart, liver, and kidney by either RT-PCR or X-gal staining. The X-gal staining-positive cells were observed at or near the luminal surface in the aortic segments only in contact with the stent graft and the ingrowth tissues within stent grafts. Immunohistochemical studies suggested that the LacZ-positive cells were mainly the neointimal α-smooth-muscle actin-positive cells and macrophages. The extent of the transgene expression was dependent on the quantity of the plasmid DNA loaded onto the stent graft (10.0 mg/mL plasmid vs 1.0 mg/mL plasmid, P < .01 and 10.0 mg/mL plasmid vs 0.1 mg/mL plasmid, P < .05). LacZ mRNA expression was maximal at day 1 and declined at day 7 (P < .05) but was still detectable.ConclusionPlasmid-loaded CG hydrogel-coated stent graft is a promising vehicle for local transgene delivery to the aortic wall and offers the possibility of transduction of therapeutic genes into the vascular wall.Clinical RelevancePlasmid-loaded cationized gelatin (CG) hydrogel-coated stent graft offers the possibility of transduction of therapeutic genes into the vascular wall. It may become relevant in interventional radiology in the future for inducing the regression of aortic aneurysm or treatment of atherosclerosis and improving graft function by facilitating the biologic healing between the aorta and graft.

PURPOSE To evaluate the local reaction of the aortic wall induced by basic fibroblast growth factor (bFGF) released from a gelatin hydrogel coated on the outer surface of a stent-graft for the purpose of biological fixation. METHODS A total of 18 nitinol-based, polyester-covered stent-grafts were implanted in 6 porcine aortas for 1 month. The implanted stent-grafts were divided into 3 groups: the control group (uncoated), the hydrogel group (coated with hydrogel containing water), and the bFGF group (coated with hydrogel containing bFGF). After stent-graft implantation, the results of intravascular ultrasound (IVUS) and qualitative and quantitative microscopic examinations were compared among the groups. RESULTS In the bFGF group, a thin white lamellar tissue was observed on IVUS images. Significantly more new intimal tissue formation was observed in all the bFGF group animals than in the other 2 groups, and alpha smooth muscle (SM) actin-positive cells (alphaSMCs) were detected in this new tissue. The alphaSMCs within the fabric of tightly woven grafts were significantly more abundant in the bFGF group than in the other groups. CONCLUSION The local controlled release of bFGF from the stent-graft significantly accelerated the proliferation of new intimal tissue between the aorta and the stent-graft and within the graft materials. These findings suggest that a graft can be fixed biologically to the aortic wall, which may contribute to the shrinkage of aneurysms following stent-grafting.

To evaluate the clinical value of various kinds of interventional techniques in the treatment of Budd-Chiari syndrome (BCS).Multiple techniques such as recanalization of the inferior vena cava (IVC) under the guidance of marker and multi-angled fluoroscopy, recanalization of the hepatic vein with a transjugular approach, PTA, Z-expandable metallic stent (Z-EMS) implantation and modified TIPSS were used to treat 103 patients with BCS.Of 103 patients with BCS, 59 patients with obstruction of IVC were treated using recanalization of IVC. Seventeen patients with hepatic vein obstruction had their hepatic veins recanalized. The rest of the patients were given other methods of interventional treatment. Of all the subjects, 101 successfully underwent their procedures, with a success rate of 98.06%; and only 2 failed to recanalization of the IVC. Fifty-three patients were treated using PTA for the first time, with a success rate of 100%. In the 48 patients undergoing Z-EMS implantation for the first time, the success rate was 95.8%. Five patients were treated with modified TIPSS. After these interventional treatments, the success rate was 100%. Two patients died 16 h and 72 h respectively after operation because of DIC and severe hemoptysis. Seventy-two patients were followed up for 1 - 94 months (with a mean of 42.3 months). The mean follow-up of a BCS patient treated with PTA was 52.1 months, resulting in a primary patent rate of 59.4% and a restenosis rate of 40.6%. The mean follow-up of BCS treated with stenting was 33.5 months, with a primary patent rate of 87.5% and a restenosis rate of 12.5%. Eight patients died 7 - 64 months after the interventional procedure.Recanalization of IVC or the hepatic vein transjugularly, PTA, Z-EMS implantation and modified TIPSS can be regarded as safe and effective micro-invasive methods in the treatment of BCS.