Alexander KC, Ikonomidis JS, Akerman AW. New Directions in Diagnostics for Aortic Aneurysms: Biomarkers and Machine Learning. J Clin Med. 2024 Jan 31;13(3):818. doi: 10.3390/jcm13030818. PMID: 38337512; PMCID: PMC10856211.
This review article presents an appraisal of pioneering technologies poised to revolutionize the diagnosis and management of aortic aneurysm disease, with a primary focus on the thoracic aorta while encompassing insights into abdominal manifestations. Our comprehensive analysis is rooted in an exhaustive survey of contemporary and historical research, delving into the realms of machine learning (ML) and computer-assisted diagnostics. This overview draws heavily upon relevant studies, including Siemens’ published field report and many peer-reviewed publications. At the core of our survey lies an in-depth examination of ML-driven diagnostic advancements, dissecting an array of algorithmic suites to unveil the foundational concepts anchoring computer-assisted diagnostics and medical image processing. Our review extends to a discussion of circulating biomarkers, synthesizing insights gleaned from our prior research endeavors alongside contemporary studies gathered from the PubMed Central database. We elucidate the prevalent challenges and envisage the potential fusion of AI-guided aortic measurements and sophisticated ML frameworks with the computational analyses of pertinent biomarkers. By framing current scientific insights, we contemplate the transformative prospect of translating fundamental research into practical diagnostic tools. This narrative not only illuminates present strides, but also forecasts promising trajectories in the clinical evaluation and therapeutic management of aortic aneurysm disease.
Alexander, K.C.; Anderson, C.W.; Agala, C.B.; Tasoudis, P.; Collins, E.N.; Ding, Y.; Blackwell, J.W.; Willcox, D.E.; Farivar, B.S.; Kibbe, M.R. Paradoxical Changes: EMMPRIN Tissue and Plasma Levels in Marfan Syndrome-related Thoracic Aortic Aneurysms. Journal of Clinical Medicine 2024, 13, 1548, doi:10.3390/jcm13061548.
Background: Thoracic aortic aneurysms (TAAs) associated with Marfan syndrome (MFS) are unique in that extracellular matrix metalloproteinase inducer (EMMPRIN) levels do not behave the way they do in other cardiovascular pathologies. EMMPRIN is shed into the circulation through the secretion of extracellular vesicles. This has been demonstrated to be dependent upon the Membrane Type-1 MMP (MT1-MMP). We investigated this relationship in MFS TAA tissue and plasma to discern why unique profiles may exist. Methods: Protein targets were measured in aortic tissue and plasma from MFS patients with TAAs and were compared to healthy controls. The abundance and location of MT1-MMP was modified in aortic fibroblasts and secreted EMMPRIN was measured in conditioned culture media. Results: EMMPRIN levels were elevated in MFS TAA tissue but reduced in plasma, compared to the controls. Tissue EMMPRIN elevation did not induce MMP-3, MMP-8, or TIMP-1 expression, while MT1-MMP and TIMP-2 were elevated. MMP-2 and MMP-9 were reduced in TAA tissue but increased in plasma. In aortic fibroblasts, EMMPRIN secretion required the internalization of MT1-MMP. Conclusions: In MFS, impaired EMMPRIN secretion likely contributes to higher tissue levels, influenced by MT1-MMP cellular localization. Low EMMPRIN levels, in conjunction with other MMP analytes, distinguished MFS TAAs from controls, suggesting diagnostic potential.
Maiocchi, S.; Collins EN.; Peterson AR.; Alexander KC.; McGlamery DJ.; Cassidy NA.; Ikonomidis JS.; Akerman AW. Plasma microrna quantification protocol. Vessel Plus. 2023, 7, 27. http://dx.doi.org/10.20517/2574-1209.2023.69
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.
Akerman AW, Alexander KC, Caranasos TG, Ikonomidis JS. Therapeutic potential of mesenchymal stem cells and their secreted extracellular vesicles in thoracic aortic aneurysm disease. J Thorac Cardiovasc Surg. 2023 Apr 20:S0022-5223(23)00334-3. doi: 10.1016/j.jtcvs.2023.03.033. Epub ahead of print. PMID: 37084818.
- Mesenchymal stem cells and their secreted vesicles have potential as therapeutics for thoracic aortic aneurysms. There are unique challenges to be overcome with innovative, standardized solutions.
Diehl JN, Ray A, Collins LB, Peterson A, Alexander KC, Boutros JG, Ikonomidis JS, Akerman AW. A standardized method for plasma extracellular vesicle isolation and size distribution analysis. PLoS One. 2023 Apr 28;18(4):e0284875. doi: 10.1371/journal.pone.0284875. PMID: 37115777; PMCID: PMC10146456.
- The following protocol describes our workflow for isolation and quantification of plasma extracellular vesicles (EVs). It requires limited sample volume so that the scientific value of specimens is maximized. These steps include isolation of vesicles by automated size exclusion chromatography and quantification by tunable resistive pulse sensing. This workflow optimizes reproducibility by minimizing variations in processing, handling, and storage of EVs. EVs have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. This standardized protocol is scalable and ensures efficient recovery of physiologically intact EVs that may be used in a variety of downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely. Absolute quantification corrects for variations in EV number and size, offering a novel method of standardization in downstream applications.
Ledford BT, Akerman AW, Sun K, Gillis DC, Weiss JM, Vang J, Willcox S, Clemons TD, Sai H, Qiu R, Karver MR, Griffith JD, Tsihlis ND, Stupp SI, Ikonomidis JS, Kibbe MR. Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms. ACS Nano. 2022 May 24;16(5):7309-7322. doi: 10.1021/acsnano.1c06258. Epub 2022 May 3. PMID: 35504018; PMCID: PMC9733406.
- 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.
Maiocchi S, Cartaya A, Thai S, Akerman AW, Bahnson E. Antioxidant Response Activating nanoParticles (ARAPas) localize to atherosclerotic plaque and locally activate the Nrf2 pathway. Biomater Sci. 2022 Mar 2;10(5):1231-1247. doi: 10.1039/d1bm01421h. PMID: 35076645; PMCID: PMC9181183.
- Atherosclerotic disease is the leading cause of death world-wide with few novel therapies available despite the ongoing health burden. Redox dysfunction is a well-established driver of atherosclerotic progression; however, the clinical translation of redox-based therapies is lacking. One of the challenges facing redox-based therapies is their targeted delivery to cellular domains of redox dysregulation. In the current study, we sought to develop Antioxidant Response Activating nanoParticles (ARAPas), encapsulating redox-based interventions, that exploit macrophage biology and the dysfunctional endothelium in order to selectively accumulate in atherosclerotic plaque. We employed flash nanoprecipitation (FNP) to synthesize bio-compatible polymeric nanoparticles encapsulating the hydrophobic Nrf2 activator drug, CDDO-Methyl (CDDOMe-ARAPas). Nuclear factor erythroid 2-related factor 2 (Nrf2)-activators are a promising class of redox-active drug molecules whereby activation of Nrf2 results in the expression of several antioxidant and cyto-protective enzymes that can be athero-protective. In this study, we characterize the physicochemical properties of CDDOMe-ARAPas as well as confirm their in vitro internalization by murine macrophages. Drug release of CDDOMe was determined by Nrf2-driven GFP fluorescence. Moreover, we show that these CDDOMe-ARAPas exert anti-inflammatory effects in classically activated macrophages. Finally, we show that CDDOMe-ARAPas selectively accumulate in atherosclerotic plaque of two widely-used murine models of atherosclerosis: ApoE−/− and LDLr−/− mice, and are capable of increasing gene expression of Nrf2-transcriptional targets in the atherosclerotic aortic arch. Future work will assess the therapeutic efficacy of intra-plaque Nrf2 activation with CDDOMe-ARAPas to inhibit atherosclerotic plaque progression. Overall, our present studies underline that targeting of atherosclerotic plaque is an effective means to enhance delivery of redox-based interventions.
Akerman AW, Jones JA, Ikonomidis JS. (2020) Pathophysiology of the Thoracic Aorta. The society of thoracic surgeon’s digital textbook of adult and congenital heart surgery. https://www.sts.org/education/sts-cardiothoracic-surgery-e-book
- Designed specifically for cardiovascular surgeons, it is the most complete and authoritative online and mobile resource for cardiothoracic surgery information in the world, continually updated by expert authors.
Akerman AW, Collins EN, Peterson A, Collins L, Harrison J, DeVaughn A, Townsend J, VanBurskik, Riopedre J, Reeds A, Oh J, Raybuck C, Jones JA, Ikonomidis JS. MiR-133a Replacement Attenuates Thoracic Aortic Aneurysm in Mice. Journal of the American Heart Association. 2021 Aug 17;10(16):e019862.
- Thoracic aortic aneurysms (TAAs) occur because of abnormal remodeling of aortic extracellular matrix and are accompanied by the emergence of proteolytically active myofibroblasts. The microRNA miR‐133a regulates cellular phenotypes and is reduced in clinical TAA specimens. This study tested the hypothesis that miR‐133a modulates aortic fibroblast phenotype, and overexpression by lentivirus attenuates the development of TAA in a murine model.
Newton ER, Akerman AW, Strassle PD, Kibbe MR. Association of Fluoroquinolones and short-term risk of aortic aneurysm development. JAMA Surgery. 2021 Mar 1;156(3):264-272.
- 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. 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.
Akerman AW, Blanding WM, Stroud RE, Nadeau EK, Mukherjee R, Ruddy JM, Zile MR, Ikonomidis JS, Jones JA. Elevated wall tension leads to reduced miR-133a in the thoracic aorta by exosome release. Journal of the American Heart Association. 2019 Jan 8; 8(1): e010332.
- Background Reduced miR‐133a was previously found to be associated with thoracic aortic (TA) dilation, as seen in aneurysm disease. Because wall tension increases with vessel diameter (Law of Laplace), this study tested the hypothesis that elevated tension led to the reduction of miR‐133a in the TA. Methods and Results Elevated tension (1.5 g; 150 mm Hg) applied to murine TA ex vivo reduced miR‐133a tissue abundance compared with TA held at normotension (0.7 g; 70 mm Hg). Cellular miR‐133a levels were reduced with biaxial stretch of isolated murine TA fibroblasts, whereas smooth muscle cells were not affected. Mechanisms contributing to the loss of miR‐133a abundance were further investigated in TA fibroblasts. Biaxial stretch did not reduce primary miR‐133a transcription and had no effect on the expression/abundance of 3 microRNA‐specific exoribonucleases. Remarkably, biaxial stretch increased exosome secretion, and exosomes isolated from TA fibroblasts contained more miR‐ Inhibition of exosome secretion prevented the biaxial stretch‐induced reduction of miR‐133a. Subsequently, 2 in vivo models of hypertension were used to determine the effect of elevated wall tension on miR‐133a abundance in the TA: wild‐type mice with osmotic pump–mediated angiotensin II infusion and angiotensin II–independent spontaneously hypertensive mice. Interestingly, the abundance of miR‐133a was decreased in TA tissue and increased in the plasma in both models of hypertension compared with a normotensive control group. Furthermore, miR‐133a was elevated in the plasma of hypertensive human subjects, compared with normotensive patients. Conclusions Taken together, these results identified exosome secretion as a tension‐sensitive mechanism by which miR‐133a abundance was reduced in TA fibroblasts.
Akerman AW, Stroud RE, Barrs RW, Grespin RT, McDonald LT, LaRue AC, Mukherjee R, Ikonomidis JS, Jones JA, Ruddy JM. Elevated Wall Tension Initiates Interleukin-6 Expression and Abdominal Aortic Dilation. Annals of Vascular Surgery. 2018;46:193-204.
- 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). 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.
Ikonomidis JS, Nadeau EK, Akerman AW, Stroud RE, Mukherjee R, Jones JA. Regulation of Membrane type-1 Matrix Metalloproteinase Activity and Intracellular Localization in Clinical Thoracic Aortic Aneurysms. Journal of Cardiovascular Surgery. 2017;153(3):537-546.
- 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-β. 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. 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.
Ruddy JM, Akerman AW, Kimbrough D, Nadeau EN, Stroud RE, Mukherjee R, Ikonomidis JS, Jones JA. Differential Hypertensive Protease Expression in the Thoracic Versus Abdominal Aorta. Journal of Vascular Surgery. 2017 Nov;66(5):1543-1552.
- 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.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.
Renaud L, Harris LG, Mani SK, Kasiganesan H, Chou JC, Baicu CF, Van Laer A, Akerman AW, Stroud RE, Jones JA, Zile MR, Menick DR. HDACs Regulate miR-133a Expression in Pressure Overload-Induced Cardiac Fibrosis. Circulation, Heart Failure. 2015 Nov;8(6):1094-104.
- MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by class I and IIb HDACs during hypertrophic remodeling. The results reveal that HDACs play a role in the regulation of pressure overload–induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in pressure overload–induced cardiac fibrosis.
Akerman AW, Mukherjee R. MicroRNAs Emerging As Mediators of Remodeling With Atrial Fibrillation. Heart Rhythm. 2013 Jul;10(7):1010-1.
- Atrial fibrillation (AF) is the most common cardiac arrhythmia and is now established as an independent risk factor for stroke.1 Moreover, a concomitant diagnosis of AF greatly complicates treatment for a number of disease processes such as diabetes and congestive heart failure. Given the recognized additional burden that AF places on the health-care system, significant research has been performed in an attempt to delineate mechanisms that contribute to AF initiation as well as progression. Understandably, there is an extensive body of research that has identified abnormalities in ionic channels/electrogenic processes that occur with AF (reviewed in References 2, 3). For example, abnormalities in the abundance of sodium, potassium, and calcium channels have been reported with AF.2, 3 Therefore, the determination of cellular mechanisms that regulate protein abundance may shed new light on the pathogenesis of AF
Eckhouse SR, Akerman AW, Logdon CB, Oelsen JM, Mukherjee R, Jones JA, Spinale FG. Differential Membrane Type-1 Matrix Metalloproteinase Substrate Processing with Ischemia-Reperfusion: Relationship to Interstitial microRNA Dynamics and Myocardial Function. Journal of Cardiovascular Surgery. 2013 Jan;145(1):267-275, 277.e1-4; discussion 275-7.
- 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. 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.
Ikonomidis JS, Ivey CR, Wheeler JB, Akerman AW, Rice A, Patel RK, Stroud RE, Shah AA, Hughes CG, Ferrari G, Jones JA. Plasma Biomarkers for Distinguishing Etiological Subtypes of Thoracic Aortic Aneurysm Disease. Journal of Thoracic and Cardiovascular Surgery. 2013 May;145(5):1326-33.
- 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. 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.