Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a localized dilation of the aorta located in the abdomen that poses a severe risk of death when ruptured. The cause of AAA is not fully understood, but degradation of medial elastin due to elastolytic matrix metalloproteinases is a key step leading to aortic dilation. Current therapeutic interventions are limited to surgical repair to prevent catastrophic rupture. Here, we report the development of injectable supramolecular nanofibers using peptide amphiphile molecules designed to localize to AAA by targeting fragmented elastin, matrix metalloproteinase 2 (MMP-2), and membrane type 1 matrix metalloproteinase. We designed four targeting peptide sequences from X-ray crystallographic data and incorporated them into PA molecules via solid phase peptide synthesis. After coassembling targeted and diluent PAs at different molar ratios, we assessed their ability to form nanofibers using transmission electron microscopy and to localize to AAA in male and female Sprague-Dawley rats using light sheet fluorescence microscopy. We found that three formulations of the PA nanofibers were able to localize to AAA tissue, but the MMP-2 targeting PA substantially outperformed the other nanofibers. Additionally, we demonstrated that the MMP-2 targeting PA nanofibers had an optimal dose of 5 mg (∼12 mg/kg). Our results show that there was not a significant difference in targeting between male and female Sprague-Dawley rats. Given the ability of the MMP-2 targeting PA nanofiber to localize to AAA tissue, future studies will investigate potential diagnostic and targeted drug delivery applications for AAA.

Association of Fluoroquinolone Use With Short-term Risk of Development of Aortic Aneurysm.

Importance: Although fluoroquinolones are commonly prescribed antibiotics in the US, recent international studies have shown an increased risk of aortic aneurysm and dissection after fluoroquinolone use, leading to US Food and Drug Administration warnings limiting use for high-risk patients. It is unclear whether these data are true for the US population and who is truly high risk. Objective: To assess aortic aneurysm and dissection risks in a heterogeneous US population after fluoroquinolone use. Design, Setting, and Participants: Prescription fills for fluoroquinolones or a comparator antibiotic from 2005 to 2017 among commercially insured individuals aged 18 to 64 years were identified in this retrospective analysis of MarketScan health insurance claims. This cohort study included 27 827 254 US adults (47 596 545 antibiotic episodes), aged 18 to 64 years, with no known previous aortic aneurysm or dissection, no recent antibiotic exposure, and no recent hospitalization. Exposures: Outpatient fill of an oral fluoroquinolone or comparator antibiotic (amoxicillin-clavulanate, azithromycin, cephalexin, clindamycin, and sulfamethoxazole-trimethoprim). Main Outcomes and Measures: The 90-day incidence of aortic aneurysm and dissection. Inverse probability of treatment weighting in Cox regression was used to estimate the association between fluoroquinolone fill and 90-day aneurysm incidence. Interaction terms were used to assess the association of known risk factors (ie, sex, age, and comorbidities) with aneurysm after fluoroquinolone use. Data analysis was performed March 2019 to May 2020. Results: Of 47 596 545 prescription fills, 9 053 961 (19%) were fluoroquinolones and 38 542 584 (81%) were comparator antibiotics. The median (interquartile range) age of adults with fluoroquinolone fills was 47 (36-57) years vs 43 (31-54) years with comparator antibiotic fills. Women comprised 61.3% of fluoroquinolone fills and 59.5% of comparator antibiotic fills. Before weighting, the 90-day incidence of newly diagnosed aneurysm was 7.5 cases per 10 000 fills (6752 of 9 053 961) after fluoroquinolones compared with 4.6 cases per 10 000 fills (17 627 of 38 542 584) after comparator antibiotics. After weighting for demographic characteristics and comorbidities, fluoroquinolone fills were associated with increased incidence of aneurysm formation (hazard ratio [HR], 1.20; 95% CI, 1.17-1.24). More specifically, compared with comparator antibiotics, fluoroquinolone fills were associated with increased 90-day incidence of abdominal aortic aneurysm (HR, 1.31; 95% CI, 1.25-1.37), iliac artery aneurysm (HR, 1.60; 95% CI, 1.33-1.91), and other abdominal aneurysm (HR, 1.58; 95% CI, 1.39-1.79), and adults were more likely to undergo aneurysm repair (HR, 1.88; 95% CI, 1.44-2.46). When stratified by age, all adults 35 years or older appeared at increased risk (18-34 years: HR, 0.99 [95% CI, 0.83-1.18]; 35-49 years: HR, 1.18 [95% CI, 1.09-1.28]; 50-64 years: HR, 1.24 [95% CI, 1.19-1.28]; P = .04). Conclusions and Relevance: This study found that fluoroquinolones were associated with increased incidence of aortic aneurysm formation in US adults. This association was consistent across adults aged 35 years or older, sex, and comorbidities, suggesting fluoroquinolone use should be pursued with caution in all adults, not just in high-risk individuals.

Elevated Wall Tension Initiates Interleukin-6 Expression and Abdominal Aortic Dilation.

BACKGROUND: Hypertension (HTN) has long been associated with abdominal aortic aneurysm (AAA) development, and these cardiovascular pathologies are biochemically characterized by elevated plasma levels of angiotensin II (AngII) as well as interleukin-6 (IL-6). A biologic relationship between HTN and AAA has not been established, however. Accordingly, the objective of this study was to evaluate whether elevated tension may initiate IL-6 production to accumulate monocyte/macrophages and promote dilation of the abdominal aorta (AA). METHODS: An IL-6 infusion model (4.36 µg/kg/day) was created utilizing an osmotic infusion pump, and after 4 weeks, AA diameter was measured by digital microscopy. The AA was then excised for CD68 immunostaining and flow cytometric analysis with CD11b and F4/80 to identify macrophages. Aortic segments from wild-type mice were suspended on parallel wires in an ex vivo tissue myograph at experimentally derived optimal tension (1.2 g) and in the presence of elevated tension (ET, 1.7 g) for 3 hr, and expression of IL-6 and monocyte chemoattractant protein-1 (MCP-1) was evaluated by quantitative polymerase chain reaction (QPCR). Isolated aortic vascular smooth muscle cells (VSMCs) were subjected to 12% biaxial cyclic stretch or held static (control) for 3 hr (n = 7), and IL-6 and MCP-1 expressions were evaluated by QPCR. RESULTS: Four-week IL-6 infusion resulted in an AA outer diameter that was 72.5 ± 5.6% (P < 0.05) greater than that of control mice, and aortic dilation was accompanied by an accumulation of macrophages in the AA medial layer as defined by an increase in CD68 + staining as well as an increase by flow cytometric quantification of CD11b+/F4/80+ cells. Wild-type AA segments did not respond to ex vivo application of ET but cyclic stretch of isolated VSMCs increased IL-6 (2.03 ± 0.3 fold) and MCP-1 (1.51 ± 0.11 fold) expression compared to static control (P < 0.05). Pretreatment with the selective STAT3 inhibitor WP1066 blunted the response in both cases. Interestingly, AngII did not stimulate expression of IL-6 and MCP-1 above that initiated by tension and again, the response was inhibited by WP1066, supporting an integral role of STAT3 in this pathway. CONCLUSIONS: An IL-6 infusion model can initiate macrophage accumulation as well as aortic dilation, and under conditions of elevated tension, this proinflammatory cytokine can be produced by aortic VSMCs. By activation of STAT3, MCP-1 is expressed to increase media macrophage abundance and create an environment susceptible to dilation. This biomechanical association between HTN and aortic dilation may allow for the identification of novel therapeutic strategies.

Differential hypertensive protease expression in the thoracic versus abdominal aorta.

BACKGROUND: Hypertension (HTN), which is a major risk factor for cardiovascular morbidity and mortality, can drive pathologic remodeling of the macro- and microcirculation. Patterns of aortic pathology differ, however, suggesting regional heterogeneity of the pressure-sensitive protease systems triggering extracellular matrix remodeling in the thoracic (TA) and abdominal aortas (AA). This study tested the hypothesis that the expression of two major protease systems (matrix metalloproteinases [MMPs] and cathepsins) in the TA and AA would be differentially affected with HTN. METHODS: Normotensive (BPN3) mice at 14-16 weeks of age underwent implantation of osmotic infusion pumps for 28-day angiotensin II (AngII) delivery (1.46 mg/kg/day; BPN3+AngII; n = 8) to induce HTN. The TA and AA were harvested to determine levels of MMP-2, MMP-9, and membrane type 1-MMP, and cathepsins S, K, and L were evaluated in age-matched BPN3 (n = 8) control and BPH2 spontaneously hypertensive mice (non-AngII pathway; n = 7). Blood pressure was monitored via CODA tail cuff plethysmography (Kent Scientific Corporation, Torrington, Conn). Quantitative real-time polymerase chain reaction and immunoblotting/zymography were used to measure MMP and cathepsin messenger RNA expression and protein abundance, respectively. Target protease values were compared within each aortic region via analysis of variance. RESULTS: Following 28 days infusion, the BPN3+AngII mice had a 17% increase in systolic blood pressure, matching that of the BPH2 spontaneously hypertensive mice (both P < .05 vs BPN3). MMP-2 gene expression demonstrated an AngII-dependent increase in the TA (P < .05), but MMP-9 was not altered with HTN. Expression of tissue inhibitor of metalloproteinases-1 was markedly increased in TA of BPN3+AngII mice, but tissue inhibitor of metalloproteinases-2 demonstrated decreased expression in the AA of both hypertensive groups (P < .05). Only cathepsin K responded to AngII-induced HTN with significant elevation in the TA of those mice, but expression of cathepsin L and cystatin C was inhibited in AA of both hypertensive groups (P < .05). Apoptotic markers were not significantly elevated in any experimental group. CONCLUSIONS: By using two different models of HTN, this study has identified pressure-dependent as well as AngII-dependent regional alterations in aortic gene expression of MMPs and cathepsins that may lead to differential remodeling responses in each of the aortic regions. Further studies will delineate mechanisms and may provide targeted therapies to attenuate down-stream aortic pathology based on demonstrated regional heterogeneity.

Regulation of membrane type-1 matrix metalloproteinase activity and intracellular localization in clinical thoracic aortic aneurysms.

OBJECTIVE: Membrane type-1 matrix metalloproteinase (MT1-MMP) is elevated during thoracic aortic aneurysm (TAA) development in mouse models, and plays an important role in the activation of matrix metalloproteinase (MMP)-2 and the release of matrix- bound transforming growth factor-ß. In this study, we tested the hypothesis that MT1-MMP is subject to protein kinase C (PKC)-mediated regulation, which alters intracellular trafficking and activity with TAAs. METHODS: Levels of MMP-2, native and phosphorylated MT1-MMP, and PKC-δ were measured in aortic tissue from patients with small TAAs (<5 cm; n = 8) and large TAAs (>6.5 cm; n = 8), and compared with values measured in normal controls (n = 8). Cellular localization of green fluorescent protein (GFP)-tagged MT1-MMP was assessed in aortic fibroblasts isolated from control and 4-week TAA mice. The effects of PKC-mediated phosphorylation on MT1-MMP cellular localization and function (active MMP-2 vs phospo-Smad2 abundance) were assessed after treatment with a PKC activator (phorbol-12-myristate-13-acetate [PMA], 100 nM) with and without a PKC-δ-specific inhibitor (röttlerin, 3 µM). RESULTS: Compared with controls, MT1-MMP abundance was increased in aortas from both TAA groups. Active MMP-2 was increased only in the large TAA group. The abundances of phosphorylated MT1-MMP and activated PKC-δ were enhanced in the small TAA group compared with the large TAA group. MT1-MMP was localized on the plasma membrane in aortic fibroblasts from control mice and in endosomes from TAA mice. Treatment with PMA induced MT1-MMP-GFP internalization, enhanced phospho-Smad2, and reduced MMP-2 activation, whereas röttlerin pretreatment inhibited these effects. CONCLUSIONS: Phosphorylation of MT1-MMP mediates its activity through directing cellular localization, shifting its role from MMP-2 activation to intracellular signaling. Thus, targeted inhibition of MT1-MMP may have therapeutic relevance as an approach to attenuating TAA development.

Plasma biomarkers for distinguishing etiologic subtypes of thoracic aortic aneurysm disease.

BACKGROUND: Thoracic aortic aneurysms (TAAs) develop through an asymptomatic process resulting in gross dilation that progresses to rupture if left undetected and untreated. If detected, patients with TAA are followed over time until the risk of rupture outweighs the risk of surgical repair. Current methodologies for tracking TAA size are limited to expensive computed tomography or magnetic resonance imaging because no acceptable population screening tools are currently available. Previous studies from this laboratory and others have identified differential protein profiles for the matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), in ascending TAA tissue from patients with bicuspid aortic valves (BAVs), versus patients with idiopathic degenerative disease and a tricuspid aortic valve (TAV). In addition, altered microRNA (miR) expression levels have also been reported in TAAs compared with normal aortic tissue. The objective of our study was to identify circulating factors within plasma that could serve as potential biomarkers for distinguishing etiologic subtypes of aneurysm disease. METHODS: Ascending TAA tissue and plasma specimens were obtained from patients with BAV (n = 21) and TAV (n = 21) at the time of surgical resection. The protein abundance of key MMPs (1, 2, 3, 8, and 9), TIMPs (1, 2, 3, and 4), and miRs (1, 21, 29a, 133a, 143, and 145) was examined using a multianalyte protein profiling system or by quantitative polymerase chain reaction, respectively. Results were compared with normal aortic tissue and plasma obtained from patients without aortic disease (n = 10). RESULTS: Significant (P < .05) differences in standardized miR-1 and miR-21 abundance between BAV and TAV aortic tissue samples and different tissue and plasma profiles of analyte differences from normal aorta where observed between the BAV and TAV groups. Linear regression analysis revealed significant linear relationships in plasma and tissue measurements only for MMP-8 and TIMP-1, TIMP-3, and TIMP-4 (P < .05). Receiver operator curve analysis revealed specific cassettes of analytes predictive of TAA disease. Relative to normal aorta, BAV proteolytic balance was significantly increased for MMP-1, MMP-2, and MMP-7, and for decreased MMP-8 and MMP-9. In contrast, TAV proteolytic balance relative to normal aorta was significantly increased only for MMP-1 and decreased for MMP-8 and MMP-9. CONCLUSIONS: Taken together, these unique data demonstrate differential plasma profiles of MMPs, TIMPs, and miRs in ascending TAA specimens from patients with BAV and TAV. These results suggest that circulating biomarkers may form the foundation for a broader platform of biomarkers capable of detecting the presence of TAA using a simple blood test and may also be useful in personalized strategies to distinguish between etiologic subtypes of TAAs in patients with aneurysm disease.

Differential membrane type 1 matrix metalloproteinase substrate processing with ischemia-reperfusion: relationship to interstitial microRNA dynamics and myocardial function.

OBJECTIVES: Membrane type 1 matrix metalloproteinase (MT1-MMP) is critical to a number of proteolytic and profibrotic events. However, upstream regulation of MT1-MMP with myocardial ischemia-reperfusion remains poorly understood. MicroRNAs regulate post-transcriptional events, and in silico mapping has identified a conserved sequence in MT1-MMP for microRNA-133a. This study tested the hypothesis that changes in microRNA-133a regulation occur with myocardial ischemia-reperfusion, which contributes to time- and region-dependent changes in MT1-MMP activity and processing of MT1-MMP substrates. METHODS: Yorkshire pigs (n = 12) underwent ischemia-reperfusion (90 minutes ischemia and 120 minutes reperfusion), where regional preload recruitable stroke work (sonomicrometry), interstitial MT1-MMP activity (microdialysis), Smad2 abundance (immunoblotting), and interstitial microRNA-133a (polymerase chain reaction) were determined within the ischemia-reperfusion and remote regions. Human left ventricular fibroblasts were transduced with microRNA-133a and anti-microRNA-133a (lentivirus) to determine the effects on MT1-MMP protein abundance. RESULTS: With ischemia-reperfusion, regional preload recruitable stroke work decreased from steady state (139 ± 20 mm Hg to 44 ± 11 mm Hg, P < .05) within the ischemia-reperfusion region. MT1-MMP activity increased in both regions. Phosphorylated Smad2 increased within the ischemia-reperfusion region. Both in vitro and in vivo interstitial levels of microRNA-133a decreased with ischemia and returned to steady-state levels with reperfusion. In vitro transduction of microRNA-133a in left ventricular fibroblasts decreased MT1-MMP levels. CONCLUSIONS: Modulation of MT1-MMP activity and microRNA-133a exportation into the myocardial interstitium occurred in the setting of acute myocardial ischemia-reperfusion. In addition, changes in microRNA-133a expression in left ventricular fibroblasts resulted in an inverse modulation of MT1-MMP abundance. Therefore, targeting of microRNA-133a represents a potentially novel means for regulating the cascade of profibrotic events after ischemia-reperfusion.