Loss Of Smooth Muscle Cells Research Articles

Matricellular protein CCN3 mitigates abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease.

Micro-RNAs in abdominal aortic aneurysms: insights from animal models and relevance to human disease.

Abdominal aortic aneurysm (AAA) is a major health concern and may be associated with high rates of mortality linked to acute complications. Diagnosis and treatment are, respectively, based on imaging and surgical techniques. Drug-based therapies are still mostly ineffective, which highlight a real unmet need. Major pathophysiological mechanisms leading to aneurysm formation involve inflammatory processes, degradation of the extracellular matrix, and loss of smooth muscle cells. However, the precise cellular and molecular pathways are still poorly understood. Recently, microRNAs have emerged as major intracellular players in a wide range of biological processes, and their stability in extracellular medium within microvesicles has led to propose them as mediators of intercellular crosstalk and as potential biomarkers and therapeutic targets in a variety of disease settings. To date, several studies have been performed to address the involvement of micro-RNAs (miRs) in aneurysm formation and complications. Here, we discuss the roles and implications of miRs in animal models and their relevance to human AAA.

Role of Interleukin-1 Signaling in a Mouse Model of Kawasaki Disease-Associated Abdominal Aortic Aneurysm.

Kawasaki disease (KD) is the most common cause of acquired cardiac disease in US children. In addition to coronary artery abnormalities and aneurysms, it can be associated with systemic arterial aneurysms. We evaluated the development of systemic arterial dilatation and aneurysms, including abdominal aortic aneurysm (AAA) in the Lactobacillus casei cell-wall extract (LCWE)-induced KD vasculitis mouse model. We discovered that in addition to aortitis, coronary arteritis and myocarditis, the LCWE-induced KD mouse model is also associated with abdominal aorta dilatation and AAA, as well as renal and iliac artery aneurysms. AAA induced in KD mice was exclusively infrarenal, both fusiform and saccular, with intimal proliferation, myofibroblastic proliferation, break in the elastin layer, vascular smooth muscle cell loss, and inflammatory cell accumulation in the media and adventitia. Il1r(-/-), Il1a(-/-), and Il1b(-/-) mice were protected from KD associated AAA. Infiltrating CD11c(+) macrophages produced active caspase-1, and caspase-1 or NLRP3 deficiency inhibited AAA formation. Treatment with interleukin (IL)-1R antagonist (Anakinra), anti-IL-1α, or anti-IL-1β mAb blocked LCWE-induced AAA formation. Similar to clinical KD, the LCWE-induced KD vasculitis mouse model can also be accompanied by AAA formation. Both IL-1α and IL-1β play a key role, and use of an IL-1R blocking agent that inhibits both pathways may be a promising therapeutic target not only for KD coronary arteritis, but also for the other systemic arterial aneurysms including AAA that maybe seen in severe cases of KD. The LCWE-induced vasculitis model may also represent an alternative model for AAA disease.

Allergic lung inflammation promotes atherosclerosis in apolipoprotein E-deficient mice

Inflammation drives asthma and atherosclerosis. Clinical studies suggest that asthmatic patients have a high risk of atherosclerosis. Yet this hypothesis remains uncertain, given that Th2 imbalance causes asthma whereas Th1 immunity promotes atherosclerosis. In this study, chronic allergic lung inflammation (ALI) was induced in mice by ovalbumin sensitization and challenge. Acute ALI was induced in mice by ovalbumin and aluminum sensitization and ovalbumin challenge. Atherosclerosis was produced in apolipoprotein E-deficient (Apoe(-/-)) mice with a Western diet. When chronic ALI and atherosclerosis were produced simultaneously, ALI increased atherosclerotic lesion size, lesion inflammatory cell content, elastin fragmentation, smooth muscle cell (SMC) loss, lesion cell proliferation, and apoptosis. Production of acute ALI before atherogenesis did not affect lesion size, but increased atherosclerotic lesion CD4(+) T cells, lesion SMC loss, angiogenesis, and apoptosis. Production of acute ALI after atherogenesis also did not change atherosclerotic lesion area, but increased lesion elastin fragmentation, cell proliferation, and apoptosis. In mice with chronic ALI and diet-induced atherosclerosis, daily inhalation of a mast cell inhibitor or corticosteroid significantly reduced atherosclerotic lesion T-cell and mast cell contents, SMC loss, angiogenesis, and cell proliferation and apoptosis, although these drugs did not affect lesion area, compared with those that received vehicle treatment. In conclusion, both chronic and acute ALI promote atherogenesis or aortic lesion pathology, regardless whether ALI occurred before, after, or at the same time as atherogenesis. Antiasthmatic medication can efficiently mitigate atherosclerotic lesion pathology.

Abstract WP205: C-reactive Protein Levels Are Associated With Cerebral Small Vessel-related Lesions

Introduction: Arteriosclerosis is characterized by the loss of smooth muscle cells and a hyalinized thickened wall, and concentric narrowing of the lumen in penetrating arteries causes cerebral small vessel disease, including lacunar infarcts, deep white matter lesions, and cerebral microbleeds mainly in the basal ganglia. Although infrequently fatal, lacunar infarcts and deep white matter lesions, which account for 30% of ischemic strokes in Japan, are associated with a high risk of recurrence and cognitive impairment. Basic studies have reported on the importance of inflammation in the pathogenesis of arteriosclerosis. Hypothesis: We investigated the hypothesis that serum high sensitive C-reactive protein (hs-CRP) levels have some effect on cerebral small vessel (CSV)-related lesions. Methods: From January 2008 to March 2013, a total of 1,362 Japanese subjects voluntarily participated in the health checkup system. We selected 519 neurologically normal subjects (284 males and 235 females) aged 29-90 years (mean, 63.5 ± 10.3 years) from these participants for inclusion. All the participants underwent MRI, and their CSV-related lesions (i.e., lacunar infarcts, cerebral microbleeds, deep white matter hyperintensity, and periventricular hyperintensity) were evaluated. The serum levels of hs-CRP were evaluated as common inflammatory markers. Results: Subjects with higher hs-CRP levels had more lacunar infarcts (p = 0.02). After adjusting for the traditional cardiovascular risk factors, higher hs-CRP levels were still associated with the presence of lacunar infarcts (odds ratio for the highest vs. the lowest tertile of hs-CRP, 3.57 [95% confidence interval: 1.30-9.80]). These associations did not change when the logarithmically transformed values for hs-CRP were included. Furthermore, subjects with higher CRP levels had more cerebral microbleeds (p = 0.03), more severe deep white matter hyperintensity (p = 0.04), and periventricular hyperintensity (p = 0.04); however, these associations were not observed after adjusting for the cardiovascular risk factors. Conclusions: Higher levels of hs-CRP were associated with lacunar infarcts. Thus, inflammatory processes may be involved in the pathogenesis of small vessel disease.

Disturbed integrin expression in the vascular media in CADASIL.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is an inherited angiopathy characterized by degeneration and loss of vascular smooth muscle cells (VSMCs) of still unknown pathomechanism. Many functions of VSMCs, such as adhesion, apoptosis, contraction, differentiation, migration, and proliferation are determined by integrins - surface adhesion receptors involved in binding and interactions between cells and extracellular matrix (ECM). Since integrins play such an important role in VSMCs biology, disturbances in their expression may influence myocytes behavior and fate in CADASIL. In this study, we focused on the most important compounds of VSMCs integrins: subunits α₄, β1, and β₃ in an attempt to elucidate their immune expression in the arterial media of CADASIL patients. The immunohistochemistry revealed adecreased expression of integrin β1 subunit (p < 0.001) but similar to the control expression of integrin subunits α₄ and β₃. Decreased β1 immunoreactivity was observed in capillary vessels, arterioles, and small arteries. The abnormal immune expression of integrin β1 subunit was found even in microvessels without microscopically noted degenerative changes, which suggests that this is an early phenomenon in CADASIL. Since integrin β1 subunit is acompound of 10 heterodimer integrin receptors, its disturbed expression may significantly influence VSMCs biology leading to myocytes degeneration and loss via anoikis - atype of apoptotic cell death due to loss or inappropriate cell adhesion to ECM.

Ursodeoxycholic Acid Attenuates Acute Aortic Dissection Formation in Angiotensin II-Infused Apolipoprotein E-Deficient Mice Associated with Reduced ROS and Increased Nrf2 Levels

Background/Aims: Acute aortic dissection (AAD) is characterized by excessive smooth muscle cell (SMC) loss, extracellular matrix (ECM) degradation and inflammation. In response to certain stimulations, oxidative stress is activated and regulates apoptosis and inflammation. Excessive apoptosis promotes aortic inflammation and degeneration, leading to AAD formation. This study aimed to clarify role of oxidative stress in the pathogenesis of AAD and whether the antioxidant ursodeoxycholic acid (UDCA) attenuates AAD formation. Methods: Angiotensin II (Ang II) was infused in 8-months male ApoE^-/- mice for one week to establish a model of AAD. UDCA (10 mg/kg/day) was administered via intragastric gavage for 3 consecutive days before AngII infusion and also during the AngII infusion for another consecutive 7 days. Results: Ang II-infusion resulted in the incidence of AAD at a rate of 35% (13/37) and UDCA markedly reduced the incidence of AAD to 16% (6/37), accompanied with reduced maximal aortic diameter measured at the suprarenal region of the abdominal aorta. Additionally, UDCA pretreatment prevented Ang II induced generations of reactive oxygen species (ROS) and apoptosis of vascular smooth muscle cells (VSMCs) both in vivo and in. vitro Mechanistically, we found UDCA markedly increased Nrf2 expression in VSMCs and prevented Ang II induced expression of NADPH subunits (p47, p67 and gp91) in Nrf2-dependent manner and rescued the activity of redox enzymes (Cu/Zn-SOD, Mn-SOD and CAT), thereby inhibiting apoptosis of VSMCs. Conclusion: These results demonstrate that UDCA prevented AAD formation by reducing apoptosis of VSMCs caused by oxidative stress in Nrf2 dependent manner and suggest that UDCA might have clinical potential to suppress AAD formation.

Development of a SMCs model from HGPS-iPS and proofs of principle

HGPS is a rare, progressive aging disease in children that leads to premature death. Vascular smooth muscle cells (SMCs) are the most affected cells in HGPS patients, although the reason for such sensitivity remains poorly understood. Induced pluripotent stem cells (iPSCs) offer an unlimited source of SMCs to study the disease. iPSCs are also an important tool to study the molecular mechanisms of the disease from a developmental point of view. In this work, we study the reasons of HGPS-SMCs vulnerability using iPSCs obtained from HGPS fibroblast patients. We have evaluated the differentiation profile of HGPS-iPSCs and normal iPSCs into SMCs. We showed that HGPS-iPSC SMCs shared similar features observed on progerin-expressing cells. We have identified and characterize drugs that prevent SMC loss. Our findings open new opportunities for the treatment of HGPS disease and diseases related to vascular ageing.

Abstract 11552: The α5β1 Integrin Mediates Insulin-like Growth Factor-1 Induction of Aortic Smooth Muscle Alpha-Actin Expression Through a PI3K/mTORC2/eIF4E Translational Mechanism

In arterial remodeling, smooth muscle cell (SMC) loss of contractile phenotype and increased proliferation may result in vessel stenosis or a greater propensity for aneurysm formation and rupture. Here, we show that insulin-like growth factor-1 (IGF-1) induces contractile phenotype of human aortic SMCs in culture, and we investigate the signal transduction mechanisms responsible using Western blot. IGF-1 induced a dose-dependent increase in protein expression of the contractile marker alpha-actin (αSMA) (2.4-fold-increase, 18hr serum-free vs. 100ng/mL IGF-1, p&lt;0.001), without affecting ACTA2 mRNA levels (real time RT-PCR, 4-18 hrs, p=NS). Moreover, IGF-1 upregulation of αSMA expression was not blocked by actinomycin D but was inhibited by cycloheximide (p&lt;0.01), thereby demonstrating posttranscriptional and translation-dependent regulation by IGF-1, respectively. Furthermore, IGF-1-induced αSMA expression was not blocked with PD98059 or rapamycin (50 or 100 nM), but was completely inhibited with LY294002 (p&lt;0.001), 500 nM rapamycin (p&lt;0.01), and Torin 1, thereby indicating that the ability of IGF-1 to induce SMC differentiation was independent of Erk1/2 and mTORC1, but dependent on PI3K- and mTORC2-signaling. A prolyl-hydroxylase-inhibitor (EDHB) or a function-blocking antibody against the α2β1 integrin (BHA2.1) had no effect on IGF-1-upregulation of αSMA expression. However, both EDHB and BHA2.1 significantly attenuated IGF-1-induced expression of the proliferation marker PCNA, thereby suggesting that collagen and the α2β1 integrin promote IGF-1-induced proliferation. Conversely, a blocking antibody against the α5β1 integrin (JBS5) did not block IGF-1-induced PCNA expression but completely inhibited IGF-1 induction of αSMA expression (p&lt;0.01) and abolished IGF-1-induced phosphorylation of a master regulator of cap-dependent protein translation, eukaryotic initiation factor-4E (eIF4E) (p&lt;0.001). Therefore, we concluded that IGF-1 induced αSMA expression via a PI3K/mTORC2/eIF4E translational mechanism that is dependent on the α5β1 integrin. Overall, these findings provide evidence that the α5β1 and α2β1 integrins mediate IGF-1-signaling to govern IGF-1-induced translational regulation of SMC phenotype.

Thrombomodulin Induces a Quiescent Phenotype and Inhibits Migration in Vascular Smooth Muscle Cells In Vitro

Loss of critical endothelial cell function and subsequent vascular smooth muscle cell (VSMC) migration is central to the pathology of injury-induced neointimal hyperplasia and recurrent stenosis. Thrombomodulin (TM), well known for its function as an endothelial surface anticoagulant, may have an unknown direct effect on VSMC physiology that would be lost after injury. Here, we examined a novel effect of TM on VSMC by testing the hypothesis that direct application of TM induces favorable changes to the morphology of VSMC and inhibits their migration. Primary human VSMC were harvested using the explant technique and used in early passage (1-4) for all experiments. Laser-scanning confocal fluorescent imaging was performed to assess the effect of soluble TM on VSMC morphology. In vitro, migration of VSMC was measured using: (1) a 4-hr modified Boyden chemotaxis assay and (2) a 24-hr electric cell-substrate impedance sensing injury migration assay. Migration experiments were conducted with VSMC exposed to increasing doses of soluble recombinant TM. Recombinant thrombin served as a positive control and serum-free media as a negative control for all experimentation. Data were analyzed using a Student's t-test or repeated measures analysis of variance where appropriate (α < 0.05). VSMC exposed to TM clearly demonstrated a quiescent morphology with organized stress fibers consistent with a quiescent, differentiated, contractile phenotype; whereas, thrombin stimulation led to an activated, dedifferentiated, synthetic phenotype. VSMC demonstrated a low, baseline level of migration in unstimulated serum-free conditions. Thrombin significantly stimulated VSMC migration as expected. TM, independent of thrombin, significantly inhibited baseline VSMC migration in a dose-response fashion. The maximal inhibition was observed at (5 μg/mL) with 70% reduction (56 ± 1.7 vs. 18 ± 3.5 cells/5 high-power fields, P = 0.0005). TM has a direct effect on VSMC resulting in a quiescent, differentiated and contractile phenotype, and inhibition of migration. This effect is independent of the presence of thrombin. These findings provide new knowledge in understanding the pathophysiology of vascular injury and support a strategy focused on restoring key endothelial function to prevent intimal hyperplasia.

Allergic Lung Inflammation Aggravates Angiotensin II-Induced Abdominal Aortic Aneurysms in Mice.

Asthma and abdominal aortic aneurysms (AAA) both involve inflammation. Patients with asthma have an increased risk of developing AAA or experiencing aortic rupture. This study tests the development of one disease on the progression of the other. Ovalbumin sensitization and challenge in mice led to the development of allergic lung inflammation (ALI). Subcutaneous infusion of angiotensin II into mice produced AAA. Simultaneous production of ALI in AAA mice doubled abdominal aortic diameter and increased macrophage and mast cell content, arterial media smooth muscle cell loss, cell proliferation, and angiogenesis in AAA lesions. ALI also increased plasma IgE, reduced plasma interleukin-5, and increased bronchioalveolar total inflammatory cell and eosinophil accumulation. Intraperitoneal administration of an anti-IgE antibody suppressed AAA lesion formation and reduced lesion inflammation, plasma IgE, and bronchioalveolar inflammation. Pre-establishment of ALI also increased AAA lesion size, lesion accumulation of macrophages and mast cells, media smooth muscle cell loss, and plasma IgE, reduced plasma interleukin-5, interleukin-13, and transforming growth factor-β, and increased bronchioalveolar inflammation. Consequent production of ALI also doubled lesion size of pre-established AAA and increased lesion mast cell and T-cell accumulation, media smooth muscle cell loss, lesion cell proliferation and apoptosis, plasma IgE, and bronchioalveolar inflammation. In periaortic CaCl2 injury-induced AAA in mice, production of ALI also increased AAA formation, lesion inflammation, plasma IgE, and bronchioalveolar inflammatory cell accumulation. This study suggests a pathological link between airway allergic disease and AAA. Production of one disease aggravates the progression of the other.

Inhibition of development of experimental abdominal aortic aneurysm by c-jun N-terminal protein kinase inhibitor combined with lysyl oxidase gene modified smooth muscle progenitor cells

Chronic inflammation, imbalance between the extracellular matrix synthesis and degradation, and loss of vascular smooth muscle cells (SMCs) contribute to the development of abdominal aortic aneurysm (AAA). The purpose of this study was to investigate the effect of the therapy with periaortic incubation of c-Jun N-terminal protein kinase inhibitor SP600125 infused from an osmotic pump and subadventitial injection of lysyl oxidase (LOX) gene modified autologous smooth muscle progenitor cells (SPCs) on treatment of AAA in a rabbit model. Obvious dilation of the abdominal aorta in the control group was caused by periaortic incubation of calcium chloride and elastase. But the progression of aortic dilation was significantly decreased after the treatment with SP600125 and LOX gene modified SPCs compared to the treatment with phosphate-buffered saline. This therapy could inhibit matrix metalloproteinases expression, enhance elastin synthesis, improve preservation of elastic laminar integrity, benefit SPCs survival and restore SMCs population. It seemed that this method might provide a novel therapeutic strategy to treat AAA.

THORACIC AORTIC DILATIONS IN PATIENTS WITH BICUSPID AORTIC VALVE DISEASE IS MARKED BY ACCELERATED SMOOTH MUSCLE CELL AGING

Individuals with a bicuspid aortic valve (BAV) are at increased risk for ascending aortic dilation and dissection. Loss of aortic medial smooth muscle cells (SMCs) and disruption of the extracellular matrix are well-recognized pathologies, but the underlying cellular mechanisms remain elusive. We tested the hypothesis that the dilated aorta in patients with BAV was marked by accelerated cellular aging. Samples of human ascending aorta were obtained from individuals with BAV undergoing thoracic aorta replacement (n=37, age 54.7±2.2, aortic diameter 4.8±0.9 cm) or patients with a tricuspid aortic valve and non-dilated aorta undergoing heart transplantation or coronary bypass procedures (n=8, age 55.3±8.1, aortic diameter 3.1±0.3 cm). Assessment of fresh aortic samples for senescence-associated β-galactosidase revealed evidence for rare medial cell senescence that was 4.2-fold more prevalent in dilated aortas (0.83±0.10%) than in non-dilated aortas (0.20±0.10%, p=0.048). Expression of p16 was abundantly detected in medial SMCs within dilated aortas (24.0±1.5%) and 3-fold more abundant than in non-dilated aortas (8.2±1.5%, p<0.0001). Interestingly, medial p16 expression did not correlate with telomere length of medial cells, as determined by quantitative and standardized PCR (p=0.35). Immunostaining for γH2A.X (phosphorylated Ser139) interestingly revealed discrete nuclear DNA double-strand breakage signals in 25.7±3.8% of medial cells in dilated aortas from patients with BAV, which was 2.3-fold higher than that found in non-dilated aortas (11.0±4.9, p=0.03). Medial cell outgrowths from BAV patients showed increased senescence-associated ß-galactosidase activity compared to control patient SMC outgrowths (38.9±2.9 vs. 19.5±1.9, p=0.0005). Furthermore, incubation of early passage SMCs with 50nM of angiotensin II for 3 days caused a 1.4-fold increase in senescence-associated ß-galactosidase expressing SMCs from BAV patients (p=0.0006), but elicited no change in senescence-associated ß-galactosidase expression in SMCs from control patients (p=0.83). These findings identify a previously unrecognized phenomenon of accelerated SMC aging in the aortas of patients with BAV, with cellular senescence and unresolved DNA breaks. Accelerated cell aging could thus be a driver of aortic wall degeneration in these patients and a potential therapeutic target.

Impaired LRP6-TCF7L2 Activity Enhances Smooth Muscle Cell Plasticity and Causes Coronary Artery Disease.

Mutations in Wnt-signaling coreceptor LRP6 have been linked to coronary artery disease (CAD) by unknown mechanisms. Here, we show that reduced LRP6 activity in LRP6(R611C) mice promotes loss of vascular smooth muscle cell (VSMC) differentiation, leading to aortic medial hyperplasia. Carotid injury augmented these effects and led to partial to total vascular obstruction. LRP6(R611C) mice on high-fat diet displayed dramatic obstructive CAD and exhibited an accelerated atherosclerotic burden on LDLR knockout background. Mechanistically, impaired LRP6 activity leads to enhanced non-canonical Wnt signaling, culminating in diminished TCF7L2 and increased Sp1-dependent activation of PDGF signaling. Wnt3a administration to LRP6(R611C) mice improved LRP6 activity, led to TCF7L2-dependent VSMC differentiation, and rescued post-carotid-injury neointima formation. These findings demonstrate the critical role of intact Wnt signaling in the vessel wall, establish a causal link between impaired LRP6/TCF7L2 activities and arterial disease, and identify Wnt signaling as a therapeutic target against CAD.

Comparative Histology of Aortic Dilatation Associated With Bileaflet Versus Trileaflet Aortic Valves

A more aggressive posture toward resection of the dilated aorta has been advocated when associated with bicuspid aortic valve (BAV), based on the notion that aortic material properties are weaker in this setting despite scant data to support or refute this position. The hypothesis that histologic abnormality reflects aortic wall strength was tested by comparing aortas from patients with BAV and trileaflet aortic valve. Resected aortas associated with BAV (n = 60) and trileaflet aortic valve (n = 24) were compared with normal diameter aortas from patients undergoing cardiac transplantation (n = 16) by five histologic criteria: elastic fiber loss (graded 0-4), smooth muscle cell loss (graded 0-4), medial proteoglycan accumulation (graded 0-3), medial fibrosis (graded 0-3), and atherosclerosis (graded 0-3). Patients with known connective tissue disorders, systemic inflammatory conditions, dissection, or prior heart surgery were excluded. Patients with BAV were a decade younger and more often had functional stenosis. The extent of elastic fiber loss, smooth muscle cell loss, medial fibrosis, and atherosclerosis was more severe in trileaflet aortic valve than BAV when considered across all diameters and when stratified to those between 4 and 5 cm. More severe histologic abnormalities associated with trileaflet aortic valve compared with BAV, especially when stratified by diameter, do not support a more aggressive approach to surgical intervention for dilatation associated with BAV. Indeed, if based on histologic diagnosis alone, our findings are suggestive that the converse might be true. Additionally, the lack of correlation between aortic diameter and histologic abnormality in the setting of BAV highlights the inadequacy of diameter alone as a criterion for aortic resection.

TRIF adaptor signaling is important in abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is characterized by inflammation, loss of smooth muscle cells (SMCs), and degradation of the extracellular matrix in the vessel wall. Innate immune receptors such as Toll-like receptors (TLRs) were recently shown to regulate immunological processes leading to the formation and progression of atherosclerotic plaques as well as to other cardiovascular pathologies. Our aim was to investigate whether blockage of TLR signaling, under the control of TIR domain-containing adaptor protein including IFN-β (TRIF), could inhibit the inflammatory response and AAA development in mice. In human AAA, an increased TLR3 and TLR4 expression in association with macrophages and T lymphocytes was demonstrated with immunohistochemical analysis. Angiotensin (Ang) II-induced aneurysm formation was significantly reduced by 30% in ApoE(-/-)Trif(-/-) mice compared to ApoE(-/-) mice. Morphologically, AngII-infused ApoE(-/-)Trif(-/-) mice had a more intact cellular and extracellular matrix while ApoE(-/-) mice infused with AngII displayed an increased medial thickness associated with aortic dissection, thrombus formation, and a more disorganized vessel wall. Gene expression analysis of the abdominal aorta revealed a profound decrease of the inflammatory genes CD68 (P<0.05), CD11b (P<0.05), and TNF-α (P<0.05) and the protease gene MMP-12 (P<0.01) in ApoE(-/-)Trif(-/-) mice compared to ApoE(-/-) mice infused with AngII. Our results suggest that signaling through TRIF is important for the inflammatory response of AngII-induced AAA and that blockage of the TRIF pathway reduces vascular inflammation and protects against AAA formation.

Abstract 669: Matricellular Protein CCN3 Mitigates the Progression of Abdominal Aortic Aneurysm

Background: Structural components within the vasculature, including extracellular matrix proteins, play an integral role in modulating vascular homeostasis in health and disease. Previous studies have identified important roles of a family of secreted extracellular matrix associated proteins termed CCN (Cyr61, Ctgf, Nov) in mediating matrix homeostasis. However, a role for this family of proteins in the initiation / progression of vascular diseases remains undetermined. Methods and Results: Herein, biochemical and genetic gain- and loss-of function studies reveal a novel role for CCN3 (a member of the CCN family) in abdominal aorta aneurysm (AAA) biology. Current studies suggest CCN3 levels are reduced in the rodent aorta following angiotensin II (AngII) infusion, findings recapitulated in human AAA tissues. To address whether CCN3 deficiency affects the development of AAA, control C57BL/6 and CCN3-/- mice (n=30 per group) were subjected to AngII infusion. In response to four-week AngII infusion, the aortas of CCN3-/- mice demonstrated exquisite sensitivity with more than seventy percent of CCN3-/- mice developing visible AAA. Pathologically, the AAA phenotype of CCN3-/- mice was manifested as follows: (1) severe disruption of medial architecture characterized by prominent elastin degradation and vessel dilation; (2) a significant loss of smooth muscle cells; (3) a profound inflammatory response as evidenced by enhanced immune cell infiltration, increase expression of inflammatory markers; (4) heightened reactive oxygen species generation. Moreover, the AAA phenotype of CCN3-/- mice was confirmed in a second model of AAA formation (elastase perfusion). Next, bone marrow transplant experiments indicate the AAA phenotype of CCN3-/- mice is intrinsic to the vasculature. Furthermore, lentiviral mediated overexpression of CCN3 is sufficient to mitigate elastase-mediate AAA progression in mice. Mechanistically, genetic and pharmacological studies suggest the ERK1/2 pathway serves as an important role in the regulation of CCN3-dependent AAA development. Conclusions: These results demonstrate CCN3 as an essential regulator of AAA biology and suggest the potential for future therapeutic strategies by manipulating CCN3 levels.

Abstract 249: Thoracic Aortic Dilation in Patients with Bicuspid Aortic Valves is Marked by Accelerated Vascular Smooth Muscle Cell Aging

Individuals with a bicuspid aortic valve (BAV) are at increased risk for ascending aortic dilation and dissection. Loss of aortic medial smooth muscle cells (SMCs) and disruption of the extracellular matrix are well-recognized pathologies, but the underlying cellular mechanisms remain elusive. We tested the hypothesis that the dilated aorta in patients with BAV was marked by accelerated cellular aging. Samples of human ascending aorta were obtained from individuals with BAV undergoing thoracic aorta replacement (n=37, age 54.7±2.2, aortic diameter 4.8±0.9 cm) or patients with a tricuspid aortic valve and non-dilated aorta undergoing heart transplantation or coronary bypass procedures (n=6, age 55.3±8.1, aortic diameter 3.1±0.3 cm). Assessment of fresh aortic samples for senescence-associated β-galactosidase revealed evidence for rare medial cell senescence that was 4.2-fold more prevalent in dilated aortas (0.83±0.10%) than in non-dilated aortas (0.20±0.10%, p=0.048). Expression of p16 was abundantly detected in medial SMCs within dilated aortas (27.0±2.1%) and 3-fold more abundant than in non-dilated aortas (8.9±1.8%, p&lt;0.0001). Interestingly, immunostaining for γH2A.X (phosphorylated Ser139) revealed discrete nuclear DNA double-strand breakage signals in 25.7±3.8% of medial cells in dilated aortas from patients with BAV, which was 2.3-fold higher than that found in non-dilated aortas (11.0±4.9, p=0.03). CONCLUSION: These findings identify a previously unrecognized phenomenon of accelerated SMC aging in the aortas of patients with BAV, with cellular senescence and unresolved DNA breaks. Accelerated cell aging could thus be a driver of aortic wall degeneration in these patients and a potential therapeutic target.

Mechanical stretch-induced endoplasmic reticulum stress, apoptosis and inflammation contribute to thoracic aortic aneurysm and dissection.

Thoracic aortic aneurysm/dissection (TAAD) is characterized by excessive smooth muscle cell (SMC) loss, extracellular matrix (ECM) degradation and inflammation. In response to certain stimuli, endoplasmic reticulum (ER) stress is activated and regulates apoptosis and inflammation. Excessive apoptosis promotes aortic inflammation and degeneration, leading to TAAD. Therefore, we studied the role of ER stress in TAAD formation. A lysyl oxidase inhibitor, 3‐aminopropionitrile fumarate (BAPN), was administrated to induce TAAD formation in mice, which showed significant SMC loss (α‐SMA level). Excessive apoptosis (TUNEL staining) and ER stress (ATF4 and CHOP), along with inflammation, were present in TAAD samples from both mouse and human. Transcriptional profiling of SMCs after mechanical stress demonstrated the expression of genes for ER stress and inflammation. To explore the causal role of ER stress in initiating degenerative signalling events and TAAD, we treated wild‐type (CHOP +/+) or CHOP −/− mice with BAPN and found that CHOP deficiency protected against TAAD formation and rupture, as well as reduction in α‐SMA level. Both SMC apoptosis and inflammation were significantly reduced in CHOP −/− mice. Moreover, SMCs isolated from CHOP −/− mice were resistant to mechanical stress‐induced apoptosis. Taken together, our results demonstrated that mechanical stress‐induced ER stress promotes SMCs apoptosis, inflammation and degeneration, providing insight into TAAD formation and progression. © 2015 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Immunolocalization of platelet-derived growth factor receptor-β (PDGFR-β) and pericytes in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

AimsCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is identified by aggregates of NOTCH3 extracellular domain (N3ECD) along capillaries and the deposition of granular osmiophilic material (GOM). We assessed the pattern of distribution of pericytes in relation to N3ECD deposits in cerebral microvessels of CADASIL subjects.MethodsWe assessed post mortem brains from (n = 50) subjects with CADASIL, cerebral small vessel disease, and similar‐age cognitively normal and older controls. Immunohistochemical and immunofluorescent staining methods were used to study the distribution and quantify immunoreactivities of the platelet‐derived growth factor receptor‐β (PDGFR‐β) (for pericytes) and microvascular markers in the frontal cortex and white matter.Results PDGFR‐β antibody stained cells typical of pericytes in capillaries and small arterioles in both the grey and white matter. PDGFR‐β reactive pericytes adopted ‘crescent’ morphology wrapped closely around capillary walls readily evident in cross‐sections. We noted considerable overlap between PDGFR‐β and N3ECD imunoreactivities in capillaries. Quantitative analysis of PDGFR‐β immunoreactivity revealed significant differences in PDGFR‐β %A in CADASIL compared with young controls (P < 0.05). PDGFR‐β %A was further positively correlated with the basement membrane marker collagen IV (r = 0.529, P = 0.009), but was not associated with GLUT‐1, the marker for endothelial cells.ConclusionsOur results suggest increased expression of PDGFR‐β immunoreactive pericytes in cerebral microvessels in CADASIL compared with similar age controls. While we cannot confirm whether PDGFR‐β‐expressing pericytes produce N3ECD and hence GOM, our findings demonstrate that up‐regulation of pericyte‐like cells is associated with microvascular changes, including loss of vascular smooth muscle cells in CADASIL.