Human Aortic Aneurysm Research Articles

Rewiring Vascular Metabolism Prevents Sudden Death due to Aortic Ruptures-Brief Report.

The goal of this study was to determine whether boosting mitochondrial respiration prevents the development of fatal aortic ruptures triggered by atherosclerosis and hypertension. Ang-II (angiotensin-II) was infused in ApoE (Apolipoprotein E)-deficient mice fed with a western diet to induce acute aortic aneurysms and lethal ruptures. We found decreased mitochondrial respiration and mitochondrial proteins in vascular smooth muscle cells from murine and human aortic aneurysms. Boosting NAD levels with nicotinamide riboside reduced the development of aortic aneurysms and sudden death by aortic ruptures. Targetable vascular metabolism is a new clinical strategy to prevent fatal aortic ruptures and sudden death in patients with aortic aneurysms.

Differences in Vasa Vasorum Distribution in Human Aortic Aneurysms and Atheromas.

The pathophysiological difference between aortic atheromas and aneurysms is unknown. We focused on the vasa vasorum (VV), which play a critical role in maintaining aortic homeostasis and are also involved in vascular diseases. We investigated the differences in VV between the atheromas and aneurysms. Human abdominal aortic samples were obtained from patients with abdominal aortic aneurysm during surgery or autopsy cases. Autopsy cases were divided into 2 groups according to atheromas. The VV were evaluated using immunohistochemical staining for von Willebrand factor. Intimal VV increased in both the atheroma and aneurysm groups, medial VV increased, and adventitial VV decreased only in the aneurysm group. We also observed that the medial VV were connected to the adventitial VV in the atheroma group and to intimal VV in the aneurysm group. We suggest the outside-in VV or inside-out VV theories. Atheroma induces hypoxia of aortic walls, and angiogenic factors might induce an increase of intimal VV derived from adventitial VV (outside-in VV). However, adventitial VV decrease induces hypoxia of aortic walls, and angiogenic factors might induce an increase of intimal VV derived from aortic lumen (inside-out VV). These differences of VV may contribute in elucidating the pathophysiology of aortic diseases.

Sildenafil (Viagra) Aggravates the Development of Experimental Abdominal Aortic Aneurysm.

BackgroundcGMP‐hydrolyzing phosphodiesterase type 5 (PDE5) regulates vascular smooth muscle cell (SMC) contraction by antagonizing cGMP‐dependent protein kinase I (PKGI)–dependent SMC relaxation. SMC contractile dysfunction is implicated in the pathogenesis of aortic aneurysm. PDE5 inhibitors have been used for treating erectile dysfunction, such as drug Viagra (sildenafil). However, a few clinical cases have reported the association of Viagra usage with aortic dissection, and reduced PDE5A expression was found in human aortic aneurysm tissues. Therefore, we aimed to investigate the effect of sildenafil on experimental abdominal aortic aneurysm (AAA), the most common form of aortic aneurysm in elderly men.Methods and ResultsAAA was induced in C57BL/6J male mice by periaortic elastase in combination with blocking elastin/collagen formation via 3‐aminopropionitrile fumarate salt for 35 days. PDE5A protein levels detected by immunostaining were significantly reduced in mouse AAA. Sildenafil application in drinking water significantly aggravated aortic wall dilation and elastin degradation with pre‐existing moderate AAA. The phosphorylation level of myosin light chain 2 at Ser19, a biochemical marker of SMC contraction, was significantly reduced by sildenafil in AAA. Proximity ligation assay further revealed that the interaction between cGMP and PKGI was significantly increased by sildenafil in AAA, suggesting an elevation of PKGI activation in AAA.ConclusionsSildenafil treatment aggravated the degradation of elastin fibers and progression of experimental AAA by dysregulating cGMP and contractile signaling in SMCs. Our findings may raise the caution of clinical usage of Viagra in aneurysmal patients.

Vascular Smooth Muscle Cells in Aortic Aneurysm: From Genetics to Mechanisms.

Aortic aneurysm, including thoracic aortic aneurysm and abdominal aortic aneurysm, is the second most prevalent aortic disease following atherosclerosis, representing the ninth‐leading cause of death globally. Open surgery and endovascular procedures are the major treatments for aortic aneurysm. Typically, thoracic aortic aneurysm has a more robust genetic background than abdominal aortic aneurysm. Abdominal aortic aneurysm shares many features with thoracic aortic aneurysm, including loss of vascular smooth muscle cells (VSMCs), extracellular matrix degradation and inflammation. Although there are limitations to perfectly recapitulating all features of human aortic aneurysm, experimental models provide valuable tools to understand the molecular mechanisms and test novel therapies before human clinical trials. Among the cell types involved in aortic aneurysm development, VSMC dysfunction correlates with loss of aortic wall structural integrity. Here, we discuss the role of VSMCs in aortic aneurysm development. The loss of VSMCs, VSMC phenotypic switching, secretion of inflammatory cytokines, increased matrix metalloproteinase activity, elevated reactive oxygen species, defective autophagy, and increased senescence contribute to aortic aneurysm development. Further studies on aortic aneurysm pathogenesis and elucidation of the underlying signaling pathways are necessary to identify more novel targets for treating this prevalent and clinical impactful disease.

MiR-143 Mediates the TLR2/NF-κB Pathway to Attenuate AngII-induced Damage to VSMCs

Abdominal aortic aneurysm (AAA) is a perilous vascular disease with inflammatory response as its main feature. It is known that the expression of miR-143 is down-regulated in the human aortic aneurysm. In this study, we investigated the effect of miR-143 on AngII-induced VSMCs to learn the potential mechanisms of miR-143 on AAA at the cellular level. The experimental results showed that the expressions of IL-1β, MCP-1, MMP9/13, TLR2, and NF-κB p65 and the percentage of TUNEL-positive cells in AngII-VSMCs were increased significantly compared with the control group. miR-143 had the opposite result. When the expression of miR-143 was up-regulated, the expression of IL-β, MCP-1, and MMP9/13 and the percentage of TUNEL-positive cells in AngII-VSMCs was suppressed. With the transfection of miR-143 over-expression plasmid, IL-1β, MCP-1, and MMP9/13 and the percentage of TUNEL-positive cells were reversed, compared to the AngII group and the AngII+oe-TLR2+miR-143 mimic group. In AngII-induced mouse VSMC, the up-regulation of the miR-143 expression could inactivate the TLR2/NF-κB pathway, thereby alleviating inflammatory response, ECM degradation, and cell apoptosis.

Incorporating fiber recruitment in hyperelastic modeling of vascular tissues by means of kinematic average.

We present a framework for considering the gradual recruitment of collagen fibers in hyperelastic constitutive modeling. An effective stretch, which is a response variable representing the true stretch at the tissue-scale, is introduced. Properties of the effective stretch are discussed in detail. The effective stretch and strain invariants derived from it are used in selected hyperelastic constitutive models to describe the tissue response. This construction is investigated in conjunction with Holzapfel-Gasser-Ogden family strain energy functions. The ensuing models are validated against a large body of uniaxial and bi-axial stress-strain response data from human aortic aneurysm tissues. Both the descriptive and the predictive capabilities are examined. The former is evaluated by the quality of constitutive fitting, and the latter is assessed using finite element simulation. The models significantly improve the quality of fitting, and reproduce the experiment displacement, stress, and strain distributions with high fidelity in the finite element simulation.

Regulation of SMC traction forces in human aortic thoracic aneurysms

Smooth muscle cells (SMCs) usually express a contractile phenotype in the healthy aorta. However, aortic SMCs have the ability to undergo profound changes in phenotype in response to changes in their extracellular environment, as occurs in ascending thoracic aortic aneurysms (ATAA). Accordingly, there is a pressing need to quantify the mechanobiological effects of these changes at single cell level. To address this need, we applied Traction Force Microscopy (TFM) on 759 cells coming from three primary healthy (AoPrim) human SMC lineages and three primary aneurysmal (AnevPrim) human SMC lineages, from age and gender matched donors. We measured the basal traction forces applied by each of these cells onto compliant hydrogels of different stiffness (4, 8, 12, 25 kPa). Although the range of force generation by SMCs suggested some heterogeneity, we observed that: 1. the traction forces were significantly larger on substrates of larger stiffness; 2. traction forces in AnevPrim were significantly higher than in AoPrim cells. We modelled computationally the dynamic force generation process in SMCs using the motor-clutch model and found that it accounts well for the stiffness-dependent traction forces. The existence of larger traction forces in the AnevPrim SMCs were related to the larger size of cells in these lineages. We conclude that phenotype changes occurring in ATAA, which were previously known to reduce the expression of elongated and contractile SMCs (rendering SMCs less responsive to vasoactive agents), tend also to induce stronger SMCs. Future work aims at understanding the causes of this alteration process in aortic aneurysms.

P-Selectin Glycoprotein Ligand-1 Deficiency Protects Against Aortic Aneurysm Formation Induced by DOCA Plus Salt.

P-selectin glycoprotein ligand-1 (PSGL-1) acts as a crucial regulator for the inflammatory cells infiltration by mediating the adhesion of leukocytes. However, the role of PSGL-1 in aortic aneurysm remains elusive. Here, we investigated the role of PSGL-1 in aortic aneurysm (AA) development. We first detected PSGL-1 expression in samples from aortic aneurysm patients and mouse AA models via western blotting, immunofluorescence, and flow cytometry, and then we used global PSGL-1 knockout mice and their wild type controls to establish an aortic aneurysm model induced by deoxycorticosterone acetate (DOCA) plus high salt (HS). The incidence, fatality rates, and the pathological changes of aortic aneurysm were analyzed in each group. The inflammation, adhesion molecules expression, and PSGL-1 mediated leukocyte-endothelial adhesion and their underlying mechanisms were explored further. Increased PSGL-1 levels were observed in human and mouse aortic aneurysm, and on leukocytes of mice treated with DOCA+HS. PSGL-1 deficiency reduced the incidence and severity of aortic aneurysm significantly, as well as decreased elastin fragmentation, collagen accumulation, and smooth muscle cells degeneration. Mechanistically, the protective effect of PSGL-1 inhibition was mediated by the reduced adhesion molecules, and the subsequently reduced leukocyte-endothelial adhesion through the NF-κB pathway, which finally led to reduced inflammatory cells infiltration and decreased inflammatory factors expression. PSGL-1 deficiency is protective against inflammatory cells migration and recruitment in the condition of AA through attenuation of leukocyte-endothelial adhesion. Inhibition of PSGL-1 may be a potential therapeutic target for the prevention and treatment of human AA.

CD248+CD8+ T lymphocytes suppress pathological vascular remodeling in human thoracic aortic aneurysms.

Aortic aneurysms are characterized by vascular inflammation, neovascularization, and extracellular matrix destruction of the aortic wall. Although experimental studies indicate a potential role of CD248 in microvessel remodeling, the functions of CD248 in human vascular pathologies remain unexplored. Here we aimed to study how CD248 interferes with pathological vascular remodeling of human aortic aneurysms. Immunofluorescent staining showed that CD248 expression was mainly localized in the CD8+ T cells infiltrating in the adventitia and media of aortic walls of patients with ascending thoracic aortic aneurysms. qPCR and immunofluorescent staining analyses revealed increased aortic CD248 expression and infiltrating CD248+CD8+ T cells in aortic aneurysms than in nonaneurysmal aortas. Flow cytometry analysis of human peripheral blood further identified a fraction of circulating CD248+ cells which was confined in the CD8+ T-cell compartment. The increased infiltrating of CD248+CD8+ T cells was coincident with reduced circulating CD248+CD8+ T cells in patients with ascending TAA when compared with patients with coronary artery diseases and healthy donors. The CD248+CD8+ T cells were characterized by upregulated IL-10 and downregulated IL-1β/INF-γ expression when compared with CD248-CD8+ T cells. Moreover, when co-cultured with human aortic endothelial cells, the CD248+CD8+ T cells not only downregulated endothelial expression of ICAM1/VCAM1 and MMP2/3 but also suppressed endothelial migration. This study shows that CD248 reduces pathological vascular remodeling via anti-inflammatory CD248+CD8+ T cells, revealing a CD248-mediated cellular mechanism against human aortic aneurysms.

MEK1/2 Inhibition in Murine Heart and Aorta After Oral Administration of Refametinib Supplemented Drinking Water.

Upregulation of the RAS-RAF-MEK-ERK-MAPK pathway is involved in the development of several human tumors, aortic aneurysms, atherosclerosis, and cardiomyopathy. Refametinib, a highly selective MEK-inhibitor, has already shown antineoplastic activity in phase II trials. Furthermore, it showed potency to attenuate aortic root growth in murine models. Current formulations of this drug however necessitate oral gavage as a delivery method for long-term studies, which is labor-intensive and induces stress and occasional injury, potentially confounding results. Therefore, we developed a novel oral administration method for refametinib. A 2-hydroxypropyl-beta-cyclodextrin (HPBCD) based drinking water preparation of refametinib was formulated, for which a selective, analytical UHPLC-UV method was developed to assess the in-use stability. Next, 16 week old male wild-type C57Bl/6J mice received either a daily dose of 50 or 75 mg/kg/day refametinib or were given regular drinking water during 7 days. In both dosage groups the refametinib plasma levels were measured (n = 10 or 7, respectively). Furthermore, pERK/total ERK protein levels were calculated in the myocardial and aortic tissue of mice receiving a daily dose of 50 mg/kg/day refametinib and untreated mice (n = 4/group). After 7 days no significant degradation of refametinib was observed when dissolved in drinking water provided that drinking bottles were protected from UV/visible light. Furthermore, a dose-dependent increase in refametinib plasma levels was found whereby active plasma levels (> 1.2 µg/mL) were obtained even in the lowest dose-group of 50 mg/kg/day. A significant reduction of pERK/total ERK protein levels compared to untreated mice was observed in aortic and myocardial tissue of mice receiving a daily dose of 50 mg/kg/day refametinib. Importantly, a relatively high mortality rate was noted in the highest dose group (n = 5). This approach provides a valid alternative oral administration method for refametinib with a reduced risk of complications due to animal manipulation and without loss of functionality, which can be implemented in future research regarding the malignant upregulation of the RAS-RAF-MEK-ERK-MAPK pathway. However, care must be taken not to exceed the toxic dose.

Role of PAR1-Egr1 in the Initiation of Thoracic Aortic Aneurysm in Fbln4-Deficient Mice.

Remodeling of the extracellular matrix plays a vital role in cardiovascular diseases. Using a mouse model of postnatal ascending aortic aneurysms (termed Fbln4SMKO), we have reported that abnormal mechanosensing led to aneurysm formation in Fbln4SMKO with an upregulation of the mechanosensitive transcription factor, Egr1 (Early growth response 1). However, the role of Egr1 and its upstream regulator(s) in the initiation of aneurysm development and their relationship to an aneurysmal microenvironment are unknown. Approach and Results: To investigate the contribution of Egr1 in the aneurysm development, we deleted Egr1 in Fbln4SMKO mice and generated double knockout mice (DKO, Fbln4SMKO; Egr1-/-). Aneurysms were prevented in DKO mice (42.8%) and Fbln4SMKO; Egr1+/- mice (26%). Ingenuity Pathway Analysis identified PAR1 (protease-activated receptor 1) as a potential Egr1 upstream gene. Protein and transcript levels of PAR1 were highly increased in Fbln4SMKO aortas at postnatal day 1 before aneurysm formed, together with active thrombin and MMP (matrix metalloproteinase)-9, both of which serve as a PAR1 activator. Concordantly, protein levels of PAR1, Egr1, and thrombin were significantly increased in human thoracic aortic aneurysms. In vitro cyclic stretch assays (1.0 Hz, 20% strain, 8 hours) using mouse primary vascular smooth muscle cells induced marked expression of PAR1 and secretion of prothrombin in response to mechanical stretch. Thrombin was sufficient to induce Egr1 expression in a PAR1-dependent manner. We propose that thrombin, MMP-9, and mechanical stimuli in the Fbln4SMKO aorta activate PAR1, leading to the upregulation of Egr1 and initiation of ascending aortic aneurysms.

Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-β-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-β-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-β-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

Inactivation of cysteine 674 in the SERCA2 accelerates experimental aortic aneurysm

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is vital to maintain intracellular calcium homeostasis. SERCA2 cysteine 674 (C674) is highly conservative and its irreversible oxidation is upregulated in human and mouse aortic aneurysms, especially in smooth muscle cells (SMCs). The contribution of SERCA2 and its redox C674 in the development of aortic aneurysm remains enigmatic. Objective: Our goal was to investigate the contribution of inactivation of C674 to the development of aortic aneurysm and the mechanisms involved. Approach and results: Using SERCA2 C674S knock-in (SKI) mouse line, in which half of C674 was substituted by serine 674 (S674) to represent partial irreversible oxidation of C674 in aortic aneurysm, we found that in aortic SMCs the replacement of C674 by S674 resulted in SMC phenotypic modulation. In SKI SMCs, the increased intracellular calcium activated calcium-dependent calcineurin, which promoted the nuclear translocation of nuclear factor of activated T-lymphocytes (NFAT) and nuclear factor kappa-B (NFκB), while inhibition of calcineurin blocked SMC phenotypic modulation. Besides, the replacement of C674 by S674 accelerated angiotensin II-induced aortic aneurysm. Conclusions: Our results indicate that the inactivation of C674 by causing the accumulation of intracellular calcium to activate calcineurin-mediated NFAT/NFκB pathways, resulted in SMC phenotypic modulation to accelerate aortic aneurysm, which highlights the importance of C674 redox state in the development of aortic aneurysms.

Divergent effects of canonical and non-canonical TGF-β signalling on mixed contractile-synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms.

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF‐β driven via Smad (canonical) and ERK (non‐canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF‐β‐driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF‐β‐dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF‐β‐dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a ‘mixed’ contractile‐synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.

Selective and marked decrease of complement receptor C5aR2 in human thoracic aortic aneurysms: a dysregulation with potential inflammatory effects

ObjectiveThe aetiology of thoracic aortic aneurysm (TAA) is largely unknown, but inflammation is likely to play a central role in the pathogenesis. In this present study, we aim to investigate...

Mapping Semaphorins and Netrins in the Pathogenesis of Human Thoracic Aortic Aneurysms.

Thoracic aortic aneurysm (TAA) is a complex life-threatening disease characterized by extensive extracellular matrix (ECM) fragmentation and persistent inflammation, culminating in a weakened aorta. Although evidence suggests defective canonical signaling pathways in TAA, the full spectrum of mechanisms contributing to TAA is poorly understood, therefore limiting the scope of drug-based treatment. Here, we used a sensitive RNA sequencing approach to profile the transcriptomic atlas of human TAA. Pathway analysis revealed upregulation of key matrix-degrading enzymes and inflammation coincident with the axonal guidance pathway. We uncovered their novel association with TAA and focused on the expression of Semaphorins and Netrins. Comprehensive analysis of this pathway showed that several members were differentially expressed in TAA compared to controls. Immunohistochemistry revealed that Semaphorin4D and its receptor PlexinB1, similar to Netrin-1 proteins were highly expressed in damaged areas of TAA tissues but faintly detected in the vessel wall of non-diseased sections. It should be considered that the current study is limited by its sample size and the use of internal thoracic artery as control for TAA for the sequencing dataset. Our data determines important neuronal regulators of vascular inflammatory events and suggest Netrins and Semaphorins as potential key contributors of ECM degradation in TAA.

Vascular Smooth Muscle Cell Plasticity and Autophagy in Dissecting Aortic Aneurysms.

Objective- Recent studies suggested the occurrence of phenotypic switching of vascular smooth muscle cells (VSMCs) during the development of aortic aneurysm (AA). However, lineage-tracing studies are still lacking, and the behavior of VSMCs during the formation of dissecting AA is poorly understood. Approach and Results- We used multicolor lineage tracing of VSMCs to track their fate after injury in murine models of Ang II (angiotensin II)-induced dissecting AA. We also addressed the direct impact of autophagy on the response of VSMCs to AA dissection. Finally, we studied the relevance of these processes to human AAs. Here, we show that a subset of medial VSMCs undergoes clonal expansion and that VSMC outgrowths are observed in the adventitia and borders of the false channel during Ang II-induced development of dissecting AA. The clonally expanded VSMCs undergo phenotypic switching with downregulation of VSMC differentiation markers and upregulation of phagocytic markers, indicative of functional changes. In particular, autophagy and endoplasmic reticulum stress responses are activated in the injured VSMCs. Loss of autophagy in VSMCs through deletion of autophagy protein 5 gene ( Atg5) increases the susceptibility of VSMCs to death, enhances endoplasmic reticulum stress activation, and promotes IRE (inositol-requiring enzyme) 1α-dependent VSMC inflammation. These alterations culminate in increased severity of aortic disease and higher incidence of fatal AA dissection in mice with VSMC-restricted deletion of Atg5. We also report increased expression of autophagy and endoplasmic reticulum stress markers in VSMCs of human dissecting AAs. Conclusions- VSMCs undergo clonal expansion and phenotypic switching in Ang II-induced dissecting AAs in mice. We also identify a critical role for autophagy in regulating VSMC death and endoplasmic reticulum stress-dependent inflammation with important consequences for aortic wall homeostasis and repair.

Rapamycin attenuates a murine model of thoracic aortic aneurysm by downregulating the miR-126–3p mediated activation of MAPK/ERK signalling pathway

Thoracic aortic aneurysm (TAA) is fatal diseases, which leads to aortic rupture and sudden death. Blood pressure-lowering drugs are ineffective for most of the patients. Our previous study demonstrated the inhibition of endothelial secreted miR-126–3p by rapamycin ameliorate the aneurysmal phenotype of smooth muscle cells (SMCs) in vitro. Hence, this study aimed to evaluate the modulation and mechanism of miR-126–3p in a murine model of TAA (Fbn1C1039G/+). Our results showed that noticeable disturbed flow (DF) was observed in the aorta of Fbn1C1039G/+ mice, and the expression of miR-126–3p was significantly increased under the DF in the cell chamber. This finding was also confirmed by tests in the corresponding DF area of the human aortic aneurysm tissue. Constant rapamycin administration significantly ameliorates the incidence and severity of Fbn1C1039G/+ mice characterized by decreased aortic media degradation, macrophage infiltration and MMP2/9 expression in the aortic wall. Mechanistic studies showed that rapamycin attenuates TAA progression by inhibiting miR-126–3p through ERK1/2 inactivation.

Mechanical stress alters the expression of calcification-related genes in vascular interstitial and endothelial cells.

Vascular wall calcification is a major pathophysiological component of atherosclerotic disease with many similarities to osteogenesis. Mechanical stress of the vascular wall may theoretically contribute to the proliferative processes by endothelial and interstitial cells. The aim of the study was to investigate the effect of mechanical stress on the expression of some calcification-related genes in primary human endothelial and interstitial cells, and how endothelial cells may stimulate the fibroblast and smooth muscle cells. Human umbilical vein endothelial and interstitial cells were subjected to cyclic stretch using a FlexCell® bioreactor, and interstitial cells were also subjected to tensile strain in cultures embedded in 3-dimensional collagen gels. The medium from endothelial cells was used to stimulate the gel-cultured interstitial cells, or the endothelium was sown directly on top. For comparison, human endothelial and smooth muscle cells were isolated from aortic wall fragments of patients with and without the aortic aneurysm. The expression of genes was measured using quantitative PCR. Four hours of cyclic stretch applied to cultured endothelial cells upregulated the mRNA expression of bone morphogenetic protein 2 (BMP-2), a major procalcific growth factor. When applied to a 3-dimensional culture of vascular interstitial cells, the medium from prestretched endothelial cells decreased the expression of BMP-2 and periostin mRNA in the fibroblasts. The static tension in gel-cultured interstitial cells upregulated BMP-2 mRNA expression. The addition of endothelial cells on the top of this culture also reduced mRNA of anticalcific genes, periostin and osteopontin. Similar changes were observed in smooth muscle cells from human aortic aneurysms compared to cells from the healthy aorta. Aortic aneurysm endothelial cells also showed an increased expression of BMP-2 mRNA. Endothelial cells respond to mechanical stress by upregulation of pro-osteogenic factor BMP-2 mRNA and modulate the expression of other osteogenic factors in vascular interstitial cells. Endothelial cells may, thus, contribute to vascular calcification when exposed to mechanical stress.

Medial Hypoxia and Adventitial Vasa Vasorum Remodeling in Human Ascending Aortic Aneurysm.

Human ascending aortic aneurysms characteristically exhibit cystic medial degeneration of the aortic wall encompassing elastin degeneration, proteoglycan accumulation and smooth muscle cell loss. Most studies have focused on the aortic media and there is a limited understanding of the importance of the adventitial layer in the setting of human aneurysmal disease. We recently demonstrated that the adventitial ECM contains key angiogenic factors that are downregulated in aneurysmal aortic specimens. In this study, we investigated the adventitial microvascular network (vasa vasorum) of aneurysmal aortic specimens of different etiology and hypothesized that the vasa vasorum is disrupted in patients with ascending aortic aneurysm. Morphometric analyses of hematoxylin and eosin-stained human aortic cross-sections revealed evidence of vasa vasorum remodeling in aneurysmal specimens, including reduced density of vessels, increased lumen area and thickening of smooth muscle actin-positive layers. These alterations were inconsistently observed in specimens of bicuspid aortic valve (BAV)-associated aortopathy, while vasa vasorum remodeling was typically observed in aneurysms arising in patients with the morphologically normal tricuspid aortic valve (TAV). Gene expression of hypoxia-inducible factor 1α and its downstream targets, metallothionein 1A and the pro-angiogenic factor vascular endothelial growth factor, were down-regulated in the adventitia of aneurysmal specimens when compared with non-aneurysmal specimens, while the level of the anti-angiogenic factor thrombospondin-1 was elevated. Immunodetection of glucose transporter 1 (GLUT1), a marker of chronic tissue hypoxia, was minimal in non-aneurysmal medial specimens, and locally accumulated within regions of elastin degeneration, particularly in TAV-associated aneurysms. Quantification of GLUT1 revealed elevated levels in the aortic media of TAV-associated aneurysms when compared to non-aneurysmal counterparts. We detected evidence of chronic inflammation as infiltration of lymphoplasmacytic cells in aneurysmal specimens, with a higher prevalence of lymphoplasmacytic infiltrates in aneurysmal specimens from patients with TAV compared to that of patients with BAV. These data highlight differences in vasa vasorum remodeling and associated medial chronic hypoxia markers between aneurysms of different etiology. These aberrations could contribute to malnourishment of the aortic media and could conceivably participate in the pathogenesis of thoracic aortic aneurysm.