Medial Smooth Muscle Cell Loss Research Articles

Biological differences of three paclitaxel- and sirolimus-coated balloons on coronary lesions in a rabbit model.

Drug-coated balloons (DCBs) are important treatment options for coronary artery disease; however, randomised controlled trials comparing various DCB technologies are sparse, and further investigations are needed. This preclinical study aimed to histologically and biologically compare the drug effects and safety of a low-dose paclitaxel-coated DCB (PCB; AGENT), a regular-dose PCB (SeQuent Please NEO) and a sirolimus-coated DCB (SCB; MagicTouch). The DCBs were inflated in the healthy iliac arteries of 18 rabbits, which were euthanised after 28 days. The treated iliac arteries and distal skeletal muscles were histopathologically evaluated, and drug concentrations were measured. In the histopathological evaluation, the medial smooth muscle cell loss score regarding depth, an indicator of drug efficacy, was significantly higher with AGENT and SeQuent Please NEO than with MagicTouch (4.0 [3.6-4.0] vs 3.7 [3.7-4.0] vs 2.2 [2.0-2.4]), with significant differences in comparisons between AGENT and MagicTouch (p<0.01) and between SeQuent Please NEO and MagicTouch (p<0.01). AGENT and SeQuent Please NEO showed comparable drug concentrations in the treated artery (p=0.61). In contrast, the drug concentrations in distal skeletal muscles were the highest for MagicTouch, followed by SeQuent Please NEO and AGENT (28.07 [13.19-52.46] ng/mg vs 0.66 [0.22-3.76] ng/mg vs 0.25 [0.04-3.23] ng/mg, respectively). This study demonstrated that PCBs might have higher efficacy and lower drug concentrations in distal skeletal muscles than the MagicTouch SCB. The efficacy of the AGENT low-dose PCB and the SeQuent Please NEO regular-dose PCB was comparable.

Local, Downstream, and Systemic Evaluation after Femoral Artery Angioplasty with Kanshas Drug-Coated Balloons In Vitro and in a Healthy Swine Model

PurposeTo evaluate the incidence of distal embolism and local vascular responses after treatment with the Kanshas drug-coated balloon (DCB) in a preclinical model. Materials and MethodsA total of 90 femoral arteries from 35 healthy swine were treated with a single-dose (×1) or triple-dose (×3) Kanshas DCB that applies the Unicoat technology with 3.2 μg/mm2 of paclitaxel. An uncoated Kanshas balloon was used as a control. The arterial wall, downstream skeletal muscle, and nontarget organs (kidneys, lungs, lymph nodes, liver, spleen, and heart) were histologically evaluated. For pharmacokinetic evaluation, a total of 40 healthy swine were treated with ×1 Kanshas DCB, and treated vessels were evaluated ex vivo with high-performance liquid chromatography-mass spectrometry. ResultsArteries treated with the Kanshas DCB showed mild proteoglycan deposition accompanied by the loss of smooth muscle cells (SMCs). These changes increased in a dose-dependent manner (medial SMC loss at 28 days in the ×1 vs ×3 groups, in depth, 1 (0.75–1.38) vs 2 (1.63–2.44); P = .0008; in circumference, 0.83 (0.67–1) vs 1.5 (1.19–1.81); P = .0071). No evidence of distal embolization in skeletal muscles (0 of 210 histological sections) and nontarget organs (0 of 345 sections) was observed. The pharmacokinetic evaluation showed high paclitaxel concentration in the treated artery (912 ng/mg, peaking at 3 minutes), which remained detectable at up to 180 days (0.04 ng/mg). ConclusionsThe Kanshas DCB showed a local drug effect in treated arteries up to 180 days with a high concentration of paclitaxel and no histological evidence of distal embolization.

Differences in Biologic Drug Effects and Distal Particulate Embolization in Three Paclitaxel-Coated Balloons for Femoropopliteal Lesions in a Rabbit Model.

A recent meta-analysis of randomized control trials demonstrated a significantly higher risk of major amputation in patients treated with drug-coated balloons (DCBs) compared with standard treatment, especially in high-dose paclitaxel-coated DCBs. Distal particulate embolization after DCB use was considered a potential cause of the higher incidence of major amputation. The current study aimed to histologically and biologically compare biologic drug effect and distal particulate embolization in 3 DCBs (a high-dose paclitaxel-coated DCB [IN.PACT Admiral] and 2 low-dose paclitaxel-coated DCBs [Ranger and Lutonix]). The DCBs were inflated in the healthy descending aortas of 18 rabbits, followed by euthanasia 28 days after the procedure. The treated descending aorta and distal skeletal muscles were histopathologically evaluated, and paclitaxel concentrations were measured. The paclitaxel concentration of the treated lesion was highest for Ranger, followed by IN.PACT and Lutonix (Ranger vs IN.PACT vs Lutonix: 1089 [745-2170] pmol/mg vs 638 [160-2075] pmol/mg vs 25 [10-304] pmol/mg, respectively; p<0.0001). In the histopathological evaluation, the angle of severe medial smooth muscle cell loss was largest for Ranger followed by IN.PACT and Lutonix (12.8 [8.0-20.4] degree vs 1.4 [1.2-5.2] degree vs 0.8 [0.5-2.5] degree, respectively), with significant differences for Ranger vs IN.PACT (p=0.007) and Ranger vs Lutonix (p=0.002). However, paclitaxel concentrations of distal skeletal muscles were lowest for Lutonix, followed by Ranger and IN.PACT (12 [1-58] pmol/mg vs 15 [13-21] pmol/mg vs 42 [19-108] pmol/mg, respectively, p<0.0001). The numbers of arteries with downstream DCB effects were highest for IN.PACT, followed by Ranger and Lutonix (Ranger vs IN.PACT vs Lutonix, 3 [3-4] vs 4 [3-7] vs 2 [1-2], respectively), which was consistent with the measured tissue paclitaxel concentrations. These findings suggest that Ranger demonstrates the strongest paclitaxel effect, as well as the second-best effect regarding distal particulate embolization, making it a good treatment option for patients with peripheral artery disease among the 3 DCBs evaluated in the current study. Further clinical head-to-head comparisons with larger numbers of patients are needed to explore which DCB is the most effective and safe treatment option.Clinical Impact:The findings of the current preclinical study suggests that Ranger demonstrates the strongest paclitaxel effect, as well as the second-best effect regarding distal particulate embolization making it a good treatment for patients with intermittent claudication and chronic limb-threatening ischemia.

Tricaprin can prevent the development of AAA by attenuating aortic degeneration

Medical therapeutic options to prevent rupture of abdominal aortic aneurysm (AAA), a critical event, must be developed. Moreover, further understanding of the process of AAA development and rupture is crucial. Previous studies have revealed that aortic hypoperfusion can induce the development of AAA, and we successfully developed a hypoperfusion-induced AAA animal model. In this study, we examined the effects of medium-chain triglycerides (MCTs), tricaprylin (C8-TG) and tricaprin (C10-TG), on hypoperfusion-induced AAA rat model. We estimated the effects of MCTs on aortic pathologies, mechanical properties of the aorta, and development of AAA. C10-TG, but not C8-TG, significantly suppressed AAA development and completely prevented the rupture. We observed that C10-TG prevented the development and rupture of AAA, but not C8-TG. Additionally, regression of AAA diameter was observed in the C10-TG group. Pathological analysis revealed C10-TG improved the hypoperfusion-induced increase in hypoxia-inducible factor-1α levels, medial smooth muscle cells (SMCs) loss, degeneration of aortic elastin and collagen fibers, and loss of aortic wall elasticity. In addition, regression of the formed AAA was observed by administration of C10-TG after AAA formation. C10-TG administration after AAA formation improved degeneration of AAA wall including degradation of aortic elastin and collagen fibers, stenosis of vasa vasorum, and loss of medial SMCs. These data suggest C10-TG can prevent AAA by attenuating aortic hypoperfusion and degeneration. Considering the clinical safety of C10-TG, C10-TG can be a promising AAA drug candidate.

ADAM (a Disintegrin and Metalloproteinase) 15 Deficiency Exacerbates Ang II (Angiotensin II)-Induced Aortic Remodeling Leading to Abdominal Aortic Aneurysm.

ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.

Smooth Muscle Cell Reprogramming in Aortic Aneurysms.

The etiology of aortic aneurysms is poorly understood, but it is associated with atherosclerosis, hypercholesterolemia, and abnormal transforming growth factor β (TGF-β) signaling in smooth muscle. Here, we investigated the interactions between these different factors in aortic aneurysm development and identified a key role for smooth muscle cell (SMC) reprogramming into a mesenchymal stem cell (MSC)-like state. SMC-specific ablation of TGF-β signaling in Apoe-/- mice on a hypercholesterolemic diet led to development of aortic aneurysms exhibiting all the features of human disease, which was associated with transdifferentiation of a subset of contractile SMCs into an MSC-like intermediate state thatgenerated osteoblasts, chondrocytes, adipocytes, and macrophages. This combination of medial SMCloss with marked increases in non-SMC aorticcell mass induced exuberant growth and dilation of the aorta, calcification and ossification of the aortic wall, and inflammation, resulting in aneurysm development.

In vivo canine study of three different coatings applied to p64 flow-diverter stents: initial biocompatibility study

BackgroundFlow-diverter stents (FDSs) have revolutionised the treatment of intracranial aneurysms. However, associated dual antiplatelet treatment is mandatory. We investigated the biocompatibility of three proprietary antithrombogenic coatings applied to FDSs.MethodsAfter Institutional Animal Care and Use Committee approval, four domestic juvenile female dogs (weight 19.9 ± 0.9 kg, mean ± standard deviation) were commenced on three different oral antiplatelet regimes: no medication (n = 1), acetylsalicylic acid (n = 2), and acetylsalicylic acid and clopidogrel (n = 1). Four p64 FDSs were randomly implanted into the subclavian, common carotid, and external carotid arteries of each dog, including both uncoated p64 stents and p64 stents coated with three different antithrombogenic hydrophilic coating (HPC). Angiography and histological examinations were performed. Wilcoxon/Kruskal-Wallis and ANOVA were used with p value < 0.05 considered as significant.ResultsMinimal inflammatory cell infiltration and no device-associated granulomatous cell inflammation were observed. No significant difference in adventitial inflammation (p = 0.522) or neointimal/medial inflammation (p = 0.384) between coated and uncoated stents as well as between the different stent groups regarding endothelial cell loss, surface fibrin/platelet deposition, medial smooth muscle cell loss, or adventitial fibrosis were found. Acute self-limiting thrombus formed on 6/16 implants (37.5%), and all of the thrombi were noted on devices implanted in the common or external carotid artery irrespective of the surface coating. Two of 12 p64 HPC-coated stents (16.7%) and 1/4 uncoated p64 stents (25%) showed severe or complete stenosis at delayed angiography.ConclusionsIn these preliminary in vivo experiments, HPC-coated p64 FDSs appeared to be biocompatible, without acute inflammation.

Biologic Drug Effect and Particulate Embolization of Drug-Eluting Stents versus Drug-Coated Balloons in Healthy Swine Femoropopliteal Arteries

PurposeTo compare the drug effect in treated vessels and downstream effects in distal skeletal muscle of drug-coated balloons (DCBs) and drug-eluting stents (DESs) in a healthy preclinical swine model. Materials and MethodsFour groups of treated iliofemoral arteries (percutaneous transluminal angioplasty [PTA]+DES, DCB+DES, DCB+bare metal stent [BMS], and DCB alone) of 12 healthy swine were assessed, with euthanasia at 30 days. Biological drug effect was evaluated using smooth muscle cell (SMC) loss score according to both depth and circumference as well as a neointimal fibrin and medial proteoglycan scores which were compared between the 4 groups. Vascular and skeletal muscle changes in regions downstream from the treated site were also assessed histologically for evidence of emboli. ResultsDESs showed greater medial SMC loss in the treated arteries irrespective of preceding DCB or PTA treatment in terms of depth (DCB+DES vs PTA+DES vs DCB+BMS vs DCB alone; median, 4.0 mm vs 3.8 mm vs 3.0 mm vs 2.2 mm; P = .009) and circumference (4.0 mm vs 3.5 mm vs 2.0 mm vs 1.2 mm, respectively; P = .007). Sections of skeletal muscles downstream from the treated arteries showed arteriolar changes of fibrinoid necrosis consistent with paclitaxel effect exclusively in the DCB groups (DCB+BMS, 26.9% of sections; DCB+DES, 14.3%; DCB alone, 19.2%; PTA+DES, 0%; P = .02). ConclusionsIn the treated arteries, irrespective of preceding DCB treatment or PTA, DES treatment showed maximum drug effects vs DCB alone or in combination with BMS placement, and there was no detrimental toxic effect in DCB-treated iliofemoral arteries before DES treatment compared with PTA before DES treatment. Downstream vascular changes were exclusively seen in groups treated with DCBs.

Safety of Zilver PTX Drug-Eluting Stent Implantation Following Drug-Coated Balloon Dilation in a Healthy Swine Model

Purpose: To compare the safety of Zilver PTX drug-eluting stents (DES) following drug-coated balloon (DCB) angioplasty or conventional balloon angioplasty (BA) in a healthy porcine iliofemoral artery model. Methods: DES implantation following DCB (DCB+DES) or BA (BA+DES) was assessed by angiography and histology in the nondiseased iliofemoral arteries of 20 animals, with sacrifice at 1, 3, and 6 months. Safety assessment compared quantitative measures of vessel integrity (eg, preservation of artery geometry, structure, and lumen dimensions; absence of aneurysm; malapposition) and histological parameters (eg, excessive inflammation). The percentage of uncovered struts could not be >30% per section and the endothelial cell loss had to be <50%. The vascular and skeletal muscle changes in the downstream regions were also assessed histologically for evidence of emboli. Results: No significant differences in safety parameters, including inflammation and endothelial cell loss, were observed between the 2 groups at all time points. Percentage of fibrin was significantly higher in DCB+DES at 3 months [20.0% (IQR 11.6, 28.4) vs BA+DES 4.2% (IQR 1.4, 9.6), respectively; p=0.04], with consistent trends between groups at all time points. Medial smooth muscle cell loss peaked at 1 month and was not statistically different between groups at any time point, although the loss was greater in the DCB+DES group. Sections with arterioles exhibiting paclitaxel-associated fibrinoid necrosis in downstream tissues were observed exclusively in the DCB group at 1 month (14.3% of sections) and 3 months (11.5%). Conclusion: This preclinical study suggests that Zilver PTX stent implantation is a safe strategy after DCB angioplasty and might be considered for patients who require stenting after DCB treatment.

Inhibition of KLF5-Myo9b-RhoA Pathway-Mediated Podosome Formation in Macrophages Ameliorates Abdominal Aortic Aneurysm.

Abdominal aortic aneurysms (AAAs) are characterized by pathological remodeling of the aortic wall. Although both increased Krüppel-like factor 5 (KLF5) expression and macrophage infiltration have been implicated in vascular remodeling, the role of KLF5 in macrophage infiltration and AAA formation remains unclear. To determine the role of KLF5 in AAA formation and macrophage infiltration into AAAs. KLF5 expression was significantly increased in human AAA tissues and in 2 mouse models of experimental AAA. Moreover, in myeloid-specific Klf5 knockout mice (myeKlf5-/- mice), macrophage infiltration, medial smooth muscle cell loss, elastin degradation, and AAA formation were markedly decreased. In cell migration and time-lapse imaging analyses, the migration of murine myeKlf5-/- macrophages was impaired, and in luciferase reporter assays, KLF5 activated Myo9b (myosin IXB) transcription by direct binding to the Myo9b promoter. In subsequent coimmunostaining studies, Myo9b was colocalized with filamentous actin, cortactin, vinculin, and Tks5 in the podosomes of phorbol 12,13-dibutyrate-treated macrophages, indicating that Myo9b participates in podosome formation. Gain- and loss-of-function experiments showed that KLF5 promoted podosome formation in macrophages by upregulating Myo9b expression. Furthermore, RhoA-GTP levels increased after KLF5 knockdown in macrophages, suggesting that KLF5 lies upstream of RhoA signaling. Finally, Myo9b expression was increased in human AAA tissues, located in macrophages, and positively correlated with AAA size. These data are the first to indicate that KLF5-dependent regulation of Myo9b/RhoA is required for podosome formation and macrophage migration during AAA formation, warranting consideration of the KLF5-Myo9b-RhoA pathway as a therapeutic target for AAA treatment.

Disruption of TGF-β signaling in smooth muscle cell prevents elastase-induced abdominal aortic aneurysm

Transforming growth factor-β (TGF-β) signaling has been significantly implicated in the pathogenesis of aneurysm, prominently the initiation and progression of abdominal aortic aneurysm (AAA). Vascular smooth muscle cell (SMC) is the principal resident cell in aortic wall and is essential for its structure and function. However, the role of TGF-β pathway in SMC for the formation of AAA remains unknown. Therefore, the goal of the present study was to investigate the effect of TGF-β pathway in SMC for AAA pathogenesis, by using a genetical smooth muscle-specific (SM-specific) TGF-βtype II receptor (Tgfbr2) disruption animal model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2f/f and MyhCre.Tgfbr2WT/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2WT/WT) underwent AAA induction by infrarenal peri-adventitial application of elastase. Fourteen days after elastase treatment, the aortas were analyzed and indicated that disruption of 1 or 2 alleles of Tgfbr2 in SMC provided markedly step-wise protection from AAA formation. And elastin degradation, medial SMC loss, macrophage infiltration, and matrix metalloproteinases (MMP) expression were all significantly reduced in Tgfbr2 deletion mice. Our study demonstrated, for the first time, that the TGF-β signaling pathway in SMC plays a critical role in AAA and disruption can prevent the aneurysm formation.

PPARδ reduces abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice by regulating extracellular matrix homeostasis and inflammatory responses

BackgroundAbdominal aortic aneurysm (AAA) is an inflammatory disorder characterized by a localized degradation of connective tissue and apoptosis of vascular smooth muscle cells. This study examined whether the ligand-activated peroxisome proliferator-activated receptor (PPAR) δ can directly antagonize angiotensin II (Ang II)-induced AAA formation in apoE-deficient mice. Methods and resultsSix-month-old male apoE-deficient mice were infused with Ang II and/or GW501516 (1.44 and 3.3mg/kg/day, respectively) via osmotic mini-pumps. At day 28, aortic size was measured and tissues were collected for analyses. Co-infusion of GW501516, an activator of PPARδ, attenuated both the incidence and the severity of Ang II-induced AAA in apoE-deficient mice. Ligand-activated PPARδ also reduced infiltration of macrophages, resulting in significant decreases in chemotactic proteins such as monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and inducible nitric oxide synthase. The anti-inflammatory effect of GW501516 was associated with the suppression of apoptotic cell death, along with the inhibition of medial smooth muscle cell loss and focal elastin destruction, which leads to a medial dissection and aortic rupture. These ameliorative effects of GW501516 on Ang II-induced aneurysm were correlated with increased expression of extracellular matrix (ECM) proteins, such as types I and III collagen, fibronectin, and elastin, along with the up-regulation of transforming growth factor-β1. In addition, ligand-activated PPARδ also increased the expression of tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3, while it strongly suppressed that of matrix metalloproteinase-2. ConclusionsPPARδ attenuates Ang II-induced AAA formation by regulating ECM homeostasis and inflammatory responses, suggesting a novel strategy for the treatment of AAA.

Tissue Uptake, Distribution, and Healing Response After Delivery of Paclitaxel via Second-Generation Iopromide-Based Balloon Coating: A Comparison With the First-Generation Technology in the Iliofemoral Porcine Model

This study sought to evaluate vascular drug uptake, distribution and response of second-generation paclitaxel coated balloon (PCB) (Cotavance, MEDRAD Interventional, Indianola, Pennsylvania) and compare it with first-generation technology, containing identical excipient and drug concentration. Original PCB technologies displayed a heterogeneous deposition of crystalline paclitaxel-iopromide inside the balloon folds, whereas second-generation PCBs consisted of more homogeneous, circumferential coatings. Paclitaxel tissue uptake was assessed in 20 iliofemoral arteries of a domestic swine. Vascular healing response was assessed in the familial hypercholesterolemic model of iliofemoral in-stent restenosis. Three weeks after bare-metal stent implantation, vascular segments were randomly revascularized with first-generation PCBs (n= 6), second-generation PCBs (n= 6), or plain balloon angioplasty (PBA) (n= 6). At28 days, angiographic and histological evaluation was performed in all treated segments. One-hour paclitaxel tissue uptake was 42% higher in the second-generation PCBs (p= 0.03) and resulted in more homogeneous segment-to-segment distribution compared with first-generation PCBs. Both angiography (percentage of diameter stenosis: second-generation 11.5 ± 11% vs. first-generation 21.9 ± 11% vs. PBA 46.5 ± 10%; p < 0.01) and histology (percentage of area stenosis: second-generation 50.5 ± 7% vs. first-generation 54.8 ± 18% vs. PBA 78.2 ± 9%; p < 0.01) showed a decrease in neointimal proliferation in both PCB groups. Histological variance of the percentage of area stenosis was lower in second-generation compared with first-generation PCBs (51.7 vs. 328.3; p=0.05). The presence of peristrut fibrin deposits (0.5 vs. 2.4; p < 0.01) and medial smooth muscle cell loss (0 vs. 1.7; p < 0.01) were lower in the second-generation compared with first-generation PCBs. In the experimental setting, second-generation PCB showed a comparable efficacy profile and more favorable vascular healing response when compared to first-generation PCB. The clinical implications of these findings require further investigation.

Vascular, downstream, and pharmacokinetic responses to treatment with a low dose drug‐coated balloon in a swine femoral artery model

This study was designed to evaluate the safety of a novel drug-coated balloon (DCB) with 2 µg/mm(2) paclitaxel and a carrier comprised of polysorbate and sorbitol in a swine femoral artery model. DCB have emerged as a therapeutic alternative in the treatment of peripheral vascular disease. The femoral arteries of 45 swine were treated with low pressure balloon inflation either 1× clinical dose (single inflation, 2 µg/mm(2) paclitaxel) or 4× dose (2 DCBs, each with 4 µg/mm(2) paclitaxel) or control (uncoated) balloons. The treated arteries, downstream vascular beds, and organs were assessed histologically at 28, 90, and 180-days. Twenty-four swine were treated with 1× dose for pharmacokinetic analysis through 30 days. Arterial tissue paclitaxel concentration was 58.8 ± 54.2 ng/mg at 1-hr and 0.3 ± 0.4 ng/mg at 30 days, whereas plasma paclitaxel could no longer be detected after 1 day. The treated arteries displayed minimal endothelial loss, fibrin deposition, and inflammation with long-term dose-dependent drug effect (medial smooth muscle cell loss) peaking at 90 days for both 1× (1.1 ± 1.4 vs. 0.0 ± 0.0, P = 0.008) and 4× dose (2.0 ± 1.5 vs. 0.0 ± 0.0, P < 0.001). In parallel, healing of the treated arteries was evident by significantly greater medial proteoglycan and collagen deposition at 180 days. No evidence of ischemia from downstream emboli or systemic toxicity was observed even for 4× DCB groups. The findings indicate desired pharmacologic levels with biologic effects at early and healing at late time points in the treated arteries, without evidence of significant downstream emboli or systemic toxicity, consistent with safety of the Lutonix DCB.

Mast Cells in Abdominal Aortic Aneurysms

Mast cells (MCs) are proinflammatory cells that play important roles in allergic responses, tumor growth, obesity, diabetes, atherosclerosis, and abdominal aortic aneurysm (AAA). Although the presence and function of MCs in atherosclerotic lesions have been thoroughly studied in human specimens, in primary cultured vascular cells, and in atherosclerosis in animals, their role in AAA was recognized only recently. Via multiple activation pathways, MCs release a spectrum of mediators — including histamine, inflammatory cytokines, chemokines, growth factors, proteoglycans, and proteases — to activate neighboring cells, degrade extracellular matrix proteins, process latent bioactive molecules, promote angiogenesis, recruit additional inflammatory cells, and stimulate vascular cell apoptosis. These activities associate closely with medial elastica breakdown, medial smooth-muscle cell loss and thinning, outer media and adventitia inflammation, aortic wall expansion, endothelium erosion, and eventual rupture and thrombosis. Experimental animal AAA models and MC reconstitution technique allowed examination of a direct role of MCs in AAA pathogenesis, and identification of the exact role of each MC-derived molecule. Genetic deficiency of MCs, pharmacological inhibition of MC degranulation, absence of MC-specific chymase and tryptase, or inhibition of these proteases, all effectively attenuated or abolished experimental AAA growth. The role of MCs have been reproduced in humans in several case-control studies, and two cohort studies showing the systemic level of MC specific chymase and tryptase is associated with aneurysmal growth rate, need for later aneurysmal repair and even overall mortality. These observations offer new opportunities to prevent or slow AAA growth in humans, and specific antimastcell drugs inhibiting degranulation of MCs have been used for especially allergic diseases for decades. It clearly calls for randomized clinical trials as no medical treatment to inhibit AAA progression so far have shown to be efficient.

Chemokine (C-C motif) receptor 2 mediates mast cell migration to abdominal aortic aneurysm lesions in mice

Mast cells participate importantly in abdominal aortic aneurysms (AAAs) by releasing inflammatory cytokines to promote vascular cell protease expression and arterial wall remodelling. Mast cells accumulate in AAA lesions during disease progression, but the exact chemokines by which mast cells migrate to the site of vascular inflammation remain unknown. This study tested the hypothesis that mast cells use chemokine (C-C motif) receptor 2 (CCR2) for their accumulation in experimental mouse AAA lesions. We generated mast cell and apolipoprotein E double-deficient (Apoe(-/-)Kit(W-sh/W-sh)) mice and found that they were protected from angiotensin II (Ang II) chronic infusion-induced AAAs compared with Apoe(-/-) littermates. Using bone-marrow derived mast cells (BMMC) from Apoe(-/-) mice and CCR2 double-deficient (Apoe(-/-)Ccr2(-/-)) mice, we demonstrated that Apoe(-/-)Kit(W-sh/W-sh) mice receiving BMMC from Apoe(-/-)Ccr2(-/-) mice, but not those from Apoe(-/-) mice, remained protected from AAA formation. Adoptive transfer of BMMC from Apoe(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice also increased lesion content of macrophages, T cells, and MHC class II-positive cells; there was also increased apoptosis, angiogenesis, cell proliferation, elastin fragmentation, and medial smooth muscle cell loss. In contrast, adoptive transfer of BMMC from Apoe(-/-)Ccr2(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice did not affect these variables. The increased AAA formation and associated lesion characteristics in Apoe(-/-)Kit(W-sh/W-sh) mice after receiving BMMC from Apoe(-/-) mice, but not from Apoe(-/-)Ccr2(-/-) mice, suggests that mast cells use CCR2 as the chemokine receptor for their recruitment in Ang II-induced mouse AAA lesions.

Comparison of the SYNERGY with the PROMUS (XIENCE V) and bare metal and polymer-only Element control stents in porcine coronary arteries

This study evaluated vascular compatibility of the novel platinum chromium alloy Element stent platform delivering abluminal everolimus from a poly-lactide-co-glycolide bioabsorbable polymer (SYNERGY stent), currently undergoing clinical trial, compared with the PROMUS (XIENCE V) and bare metal and polymer-only Element stents. Stents (n=161) were implanted one per coronary artery in 72 swine at a stent-to-artery ratio of 1.1:1. Similar numbers of each device group were explanted at each of 30, 90, 180, and 360 days (except no PROMUS (XIENCE V) stent at 360 days) for pathological analysis. There was no stent thrombosis, myocardial infarction, or strut fractures in any group. Vascular response was similar between the SYNERGY and PROMUS (XIENCE V) stents, with no thrombi and complete endothelialisation on both scanning electron microscopy and histology at 30, 90 and 180 days. There were no significant differences for the morphologic parameters of luminal thrombus, endothelial cell coverage, strut tissue coverage, inflammation, internal elastic lamina (IEL) disruption, external elastic lamina (EEL) disruption and medial smooth muscle cell loss across device groups or between time points, but there was mild but greater (p<0.0001) para-strut fibrin at 30 days for both drug-eluting stents (DES) compared with the bare and polymer-only controls; this difference completely dissipated by 90 days. Inflammation was predominantly minimal to mild for all device types. No morphometric parameters, including intimal thickness, stent profile-based area stenosis, and EEL area were significantly different when comparing the SYNERGY stent with the bare metal Element and polymer-only Element control stents at 90, 180 and 360 days. In this non-injured porcine coronary artery model, the bioabsorbable polymer SYNERGY stent demonstrated vascular compatibility equivalent to the PROMUS (XIENCE V) stent and to the bare metal and polymer-only Element stents.

Cyclosporine immunosuppression does not prevent the production of donor-specific antibody capable of mediating allograft vasculopathy

Late cardiac graft rejection, primarily mediated by allograft vasculopathy (AV), remains a major limitation to cardiac transplantation, even in the face of significant calcineurin inhibitor (CNI) immunosuppression. The role played by alloantibody in AV is unclear. Evidence that CNI immunosuppression suppresses CD4(+) T-cell function would suggest that antibody production and effector function would be severely limited in CNI-treated patients. In this study we examine the capacity of CNI-treated animals to develop effective alloantibody that can mediate AV. Wild-type (WT) B6 mice were alloimmunized using donor splenocytes or a fully major histocompatibility complex-mismatched allogeneic abdominal aortic graft in the presence of CNI immunosuppression (30 or 50 mg/kg/day cyclosporine A). Anti-serum was harvested and tested using complement-dependent in vitro cytotoxicity assays. Anti-serum was passively transferred to immunodeficient RAG1(-/-) recipients of allogeneic grafts. C4d deposition was quantified in the allografts from WT recipients. CNI immunosuppression did not prevent the development of alloantibody in response to either immunization method (p < 0.05). Passive transfer of anti-serum generated AV lesions in immunodeficient graft recipients and mediated complement-dependent destruction of donor cells (p < 0.05). C4d deposition was localized to the media of grafts of CNI treated animals. CNI therapy does not prevent the production of alloantibody with the capacity to mediate AV. C4d deposition in the media suggests a role for medial smooth muscle cell loss in antibody-mediated AV lesion development in our model.

Changing topographic Hox expression in blood vessels results in regionally distinct vessel wall remodeling

SummaryThe distinct topographic Hox expression patterns observed in vascular smooth muscle cells (VSMCs) of the adult cardiovascular system suggest that these transcriptional regulators are critical for maintaining region-specific physiological properties of blood vessels. To test this proposition, we expanded the vascular Hoxc11 expression domain normally restricted to the lower limbs by utilizing an innovative integrated tetracycline regulatory system and Transgelin promoter elements to induce Hoxc11 expression universally in VSMCs of transgenic mice. Ectopic Hoxc11 expression in carotid arteries, aortic arch and descending aorta resulted in drastic vessel wall remodeling involving elastic laminae fragmentation, medial smooth muscle cell loss, and intimal lesion formation. None of these alterations were observed upon induction of Hoxc11 transgene expression in the femoral artery, i.e. the natural Hoxc11 activity domain, although this vessel was greatly enlarged, comparable to the topographically restricted vascular changes seen in Hoxc11−/− mice. To begin defining Hoxc11-controlled pathways of vascular remodeling, we performed immunolabeling studies in conjunction with co-transfection and chromatin immunoprecipitation (ChIP) assays using mouse vascular smooth muscle (MOVAS) cells. The results suggest direct transcriptional control of two members of the matrix metalloproteinase (Mmp) family, including Mmp2 and Mmp9 that are known as key players in the inception and progression of vascular remodeling events. In summary, the severe vascular abnormalities resulting from the induced dysregulated expression of a Hox gene with regional vascular patterning functions suggest that proper Hox function and regulation is critical for maintaining vascular functional integrity.

Cathepsin L Activity Is Essential to Elastase Perfusion–Induced Abdominal Aortic Aneurysms in Mice

The development of abdominal aortic aneurysms (AAA) requires extensive aortic wall matrix degradation. Human AAA lesions express high levels of cathepsin L (CatL), one of the most potent mammalian elastases. Whether this protease participates directly in AAA pathogenesis, however, is unknown. We generated experimental AAA with aortic elastase perfusion in mice and established an essential role of CatL in AAA formation. After 14 days postperfusion, most wild-type (Ctsl(+/+)) mice developed AAA, but none of the CatL-deficient (Ctsl(-/-)) mice did. AAA lesion macrophage contents, CD4(+) T cell numbers, CD31(+) and laminin-5 angiogenic fragment γ2(+) microvessel numbers, and elastin fragmentation were all significantly lower in Ctsl(-/-) mice than in Ctsl(+/+) mice. While lesions from Ctsl(-/-) mice contained fewer Ki67(+) proliferating cells than did Ctsl(+/+) mice, the absence of CatL did not affect lesion apoptotic cell contents or medial smooth-muscle cell loss significantly. Mechanistic studies indicated that the absence of CatL reduced lesion chemokine monocyte chemotactic protein-1 content, macrophage and T-cell in vitro transmigration, and angiogenesis, and altered the expression and activities of matrix metalloproteinases and other cysteinyl cathepsins in inflammatory cells, vascular cells, and AAA lesions. CatL contributes to AAA formation by promoting lesion inflammatory cell accumulation, angiogenesis, and protease expression.