ATTR Amyloid Cardiomyopathy: Characterization of Extracellular Vesicles as Potential Disease Stratifiers and Prognostic Biomarkers
Conditions: Wild-type ATTR Amyloidosis, Amyloidosis Cardiac, Amyloidosis, Hereditary, Amyloidosis Transthyretin
Sponsor: University of Sao Paulo General Hospital

 
 
 
 
 
 
 
 
 
 
ATTR Amyloid Cardiomyopathy: Characterization of Extracellular 
Vesicles as Potential Stratifiers and Prognostic Biomarkers of the 
Disease 
 
 
 
 
 
Advisor: Prof. Dr. Felix Jose Alvarez Ramires 
Co-advisor: Prof. Dr. Fabio Fernandes 
Postdoctoral Fellow: Dr. Camila Rodrigues Moreno 
 
Clinical Unit of Cardiomyopathies and Aortic Diseases 
Departamento of Cardiopneumology 
School of Medicine – University of São Paulo (FMUSP) 
 
 
 
 
 
 
 
 
Sao Paulo 
2024

 
 
 
Versão 1.0 de 18/03/24                                               
Projeto InCor 
 
TABLE OF CONTENTS 
 
Pag 
1 
INTRODUCTION 
5 
2 
OBJECTIVES 
9 
 
2.1 
Primary objective 
9 
 
2.2 
Secondary objectives 
9 
3 
METHODOLOGY 
9 
 
3.1 
Study Site and Sampling 
10 
 
3.2 
Diagnosis of Transthyretin Amyloid Cardiomyopathy 
11 
 
3.3 
Inclusion Criteria 
12 
 
3.4 
Exclusion Criteria 
12 
 
3.5 
Study Design  
12 
 
3.6 
Collection and Biochemical Analysis of Biomarkers and 
Myocardial Injury 
13 
 
3.7 
Serum Preparation and Storage 
13 
 
3.8 
Isolation of Extracellular Vesicles 
13 
 
3.9 
Morphological Analysis of EVs by Transmission Electron 
Microscopy 
14 
 
3.10 
Proteomic Signature of EVs (LC/MS/MS) 
15 
 
3.11 
Quantification and Characterization by Nanosight - NTA 
(Nanoparticle Tracking Analysis) 
15 
 
3.12 
Statistical Analysis 
17 
 
3.13 
Ethical Considerations 
17 
 
3.14 
Risk and Benefit Assessment of the Clinical Protocol 
18 
 
         References 
19 
 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
5 
 
 
1. INTRODUCTION 
Amyloidosis is still considered a rare disease; however, it remains 
underdiagnosed, suggesting that its true prevalence is much higher. The etiology 
of the disease involves the deposition of amyloid proteins in different organs, 
including the heart. Recent data report a progressive increase in the prevalence 
of this disease in the United States, rising from 18 cases per 100,000 persons 
per year to 55.2 cases per 100,000 persons per year, reinforcing the concept of 
limited diagnosis1. Amyloid protein formation occurs through cleavage, 
denaturation, or overproduction of abnormal proteins, generating oligomers that 
may exhibit an antiparallel β-pleated sheet configuration, forming insoluble 
amyloid fibrils (7 to 10 nm in diameter) that accumulate in the extracellular space 
of tissues and may lead to organ dysfunction2. 
Although several tissues and organs may be affected due to systemic 
amyloidosis, cardiac involvement is the main cause of morbidity and mortality 
related to this condition3,4. Five types of amyloidosis can affect the heart: 
immunoglobulin light-chain amyloidosis (AL), transthyretin amyloidosis (TTR), 
serum amyloid A protein, apolipoprotein AI, and immunoglobulin heavy-chain 
amyloidosis. However, AL and TTR amyloidoses account for more than 95% of 
all cardiac amyloidosis cases5. Although AL amyloidosis is more aggressive, TTR 
amyloidosis tends to be diagnosed later due to its slower progression and 
because it is clinically well tolerated. As a consequence, 39% of patients are 
diagnosed at an advanced stage, presenting inability to walk, heart failure, or 
sudden cardiac death¹,6. 
Transthyretin (TTR) is a 127-amino-acid transport protein, expressed 
mainly by the liver. Under physiological conditions, it circulates as a 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
6 
 
homotetramer; however, due to genetic mutations or aging, these tetramers 
become destabilized, releasing monomeric TTR and enabling misfolding7. 
Transthyretin amyloidosis encompasses two forms of the disease: age-related 
protein degeneration (wild-type ATTR) and familial transthyretin amyloidosis 
(variant TTR)8. 
Patients with cardiac amyloidosis frequently present heart failure due to 
increased filling pressures with preserved ejection fraction, resulting from 
progressive thickening of the biventricular walls and diastolic dysfunction due to 
reduced cardiac compliance1,3,4. Despite the common clinical manifestations of 
cardiac amyloidosis, recent studies have shown that TTR-CA may present a 
distinct clinical phenotype: it occurs in older individuals, with manifestations such 
as carpal tunnel syndrome 4 to 5 years before the diagnosis of CA, small-fiber 
motor neuropathy, low-voltage electrocardiogram, atrial fibrillation, low-flow aortic 
stenosis with significant left ventricular hypertrophy, and heart failure 
disproportionate to the degree of aortic stenosis5,9,10,11. 
 
Although there are multiple consolidated diagnostic methods for TTR-CA, 
its early diagnosis remains a major challenge because its clinical symptoms 
overlap with those of other diseases12,13,14. In recent years, extracellular vesicles 
(EVs) have emerged as biomarkers for several diseases, including cancers and 
cardiovascular 
diseases, 
through 
their 
quantification 
and 
biological 
characterization15,16,17. These properties contain important information, as EVs 
participate in intercellular communication by transferring biological content in the 
form of proteins, lipids, nuclear material, miRNAs, and non-coding RNAs from 
their cells of origin. Thus, they may contribute to remote functional effects and 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
7 
 
promote homeostasis and immunity in physiological or pathological processes, 
including inflammatory responses18,19. 
According to their size, EVs are classified as microvesicles (0.1 to 1 μm) 
or exosomes (<0.1 μm) and are present in most body fluids. They can be 
protective—promoting the removal of harmful cellular components and 
supporting cell survival—or deleterious, favoring the signaling of mechanisms 
harmful to the heart, potentially contributing to heart failure development20,21,22. 
Growing evidence shows that EVs play essential roles in intercellular 
communication among cardiac tissue cells and are involved in regulating 
cardiomyocyte hypertrophy, apoptosis, and cardiac fibrosis23. These are 
pathophysiological features often modulated during cardiac remodeling in 
amyloid cardiomyopathy. 
However, although several studies have demonstrated the involvement of 
EVs in acute ischemic events and heart failure, the evaluation of EV profiles and 
characterization in TTR-CA remains poorly understood. To date, the few 
published studies on this topic have shown that patients with AL amyloidosis, who 
often present renal failure as a consequence of the deposition disease, had 
urinary exosomes carrying high amounts of light-chain oligomers compared with 
patients with nephropathies of other etiologies24,25. Another study demonstrated 
that EVs isolated from the brains of mice induced with Alzheimer’s disease 
contributed to neuronal death, the transport of neurotoxins, and the perpetuation 
of the overexpression of β-amyloid precursor protein that accumulated over 
time26. These data reinforce that, just as changes in EV concentration and 
biological 
composition 
occur 
in 
other 
cardiovascular 
diseases 
and 
cardiomyopathies, such modulation is also expected in amyloid cardiomyopathy. 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
8 
 
Conversely, there is evidence showing that protective exosomes may bind 
to non-vesicular proteins in each physiological environment, thereby reducing 
their circulating concentration. Therefore, it is evident that EVs may play an 
ambiguous role, exerting protective or deleterious effects27,28. Thus, we believe 
that EVs may serve as biomarkers of disease progression in cardiac amyloidosis. 
The rationale for this project is rooted in the underdiagnosed nature of 
cardiac amyloidosis, a condition often considered rare and usually identified only 
at advanced stages of life, when clinical symptoms have already become evident. 
In light of this reality, there is an urgent need to develop earlier diagnostic 
methods capable of detecting the disease before severe clinical outcomes occur. 
In this challenging context, the analysis of the proteomic signature present 
in extracellular vesicles emerges as a promising approach. These vesicles, 
released by cells, carry information that reflects their cellular origin and play a 
crucial role in intercellular communication. Thus, the proposed research aims to 
identify specific biomarkers present in extracellular vesicles that can be detected 
in the early stages of cardiac amyloidosis. It is expected that this approach will 
not only enable more effective therapeutic interventions, but also provide 
substantial improvements in the quality of life of individuals affected by this 
debilitating and often overlooked pathology. 
 
2. OBJECTIVE 
2.1 Primary Objective 
The primary objective of this study is to quantify and characterize serum 
extracellular vesicles (EVs) in patients with different forms of transthyretin-related 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
9 
 
amyloidosis (TTR) and to investigate their role as a biomarker of cardiac 
involvement progression. 
 
 
2.2 Secondary Objectives 
• Investigate the relationship between the quantity and characteristics of 
serum EVs and ventricular function in different study groups of patients 
with TTR-CA. 
• Evaluate the association of serum EVs with cardiac imaging findings, 
including 
cardiac 
magnetic 
resonance, 
echocardiography, 
and 
technetium-pyrophosphate scintigraphy, across the different patient 
groups. 
• Perform statistical analyses, such as linear or logistic regression, to 
identify significant predictors and assess the predictive value of EVs in 
relation to parameters of cardiac dysfunction/amyloidosis, as well as to 
investigate potential associations among these parameters. 
 
3. METHODOLOGY 
 
3.1 Study Site and Sampling 
This prospective, single-center study will be conducted at the Clinical Unit 
of Cardiomyopathy of the Heart Institute (InCor – HCFMUSP), located at the 
Hospital das Clínicas of the University of São Paulo School of Medicine 
(HCFMUSP), in São Paulo, Brazil. This center is nationally and internationally 
recognized for its excellence in the treatment of cardiac diseases and has a 
complete and adequate infrastructure for the conduct of scientific studies. 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
10 
 
Sixty patients will be selected from those treated at the Clinical Unit of 
Cardiomyopathy of InCor – HCFMUSP and will be subdivided into three distinct 
groups, as described below: 
• 
TTR-CA + DM (n=20): TTR cardiac amyloidosis with myocardial 
dysfunction; 
• 
TTR-A (n=20): TTR amyloidosis with neurological involvement; 
• 
TTR-Gen (n=10): patients who carry the genotype but do not present the 
phenotype of TTR amyloidosis; 
• 
CTL (n=20): Healthy individuals; 
 
Volunteer selection will be based on the study’s inclusion criteria. Eligible 
patients will be invited to participate and will receive a detailed explanation 
regarding the objectives, procedures, potential risks, and benefits of the study. 
Participation will be voluntary and subject to the signing of the Informed Consent 
Form (ICF) by the participant or by a close family member if the patient is 
incapacitated. The limited sample size is a direct consequence of the rarity of the 
disease under investigation.  
 
3.2 Diagnosis of Transthyretin Amyloid Cardiomyopathy 
Patients with TTR cardiac amyloidosis will be diagnosed based on a 
combination of imaging methods and clinical assessments. These methods 
include: 
• 
Electrocardiogram (ECG): For evaluation and detection of possible 
electrical abnormalities. 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
11 
 
• 
Echocardiography with Tissue Doppler and Strain Analysis: This 
technique will allow the assessment of myocardial thickening, increased 
interatrial septal thickness, enlargement and increased volume of the left 
atrium, as well as the identification of diastolic dysfunction. 
 
3.3 Inclusion Criteria 
• Adult patients aged 18 years or older; 
• Confirmed diagnosis of transthyretin-related cardiac amyloidosis (TTR-
CA), with or without myocardial dysfunction, according to established diagnostic 
criteria; 
• Willingness to comply with study procedures and requirements; 
• Ability to provide written informed consent;  
 
3.4 Exclusion Criteria 
• Presence of other significant cardiac conditions that may interfere with the 
study outcomes, such as severe coronary artery disease or major valvular 
disease; 
• Inability to provide informed consent or to participate in the required clinical 
assessments and examinations;  
 
3.5 Study Design 
After participation in the study is confirmed, the initial clinical examinations 
will be scheduled. Volunteers will be evaluated regarding their personal and 
family health history, and the variables to be collected are described in the study’s 
Case Report Form (CRF).  

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
12 
 
3.6 Collection and Biochemical Analysis of Myocardial Injury Markers 
Approximately 10 mL of whole blood will be collected from all study 
participants in dry tubes for the analysis of biochemical markers of myocardial 
injury, such as CK-MB, troponin, and BNP. The tubes will then be kept for one 
hour at 37°C to allow clot retraction. Next, the tubes will be centrifuged at 2,000 
rpm for 10 minutes, and the serum will be separated, aliquoted into microtubes, 
and stored in an ultra-freezer at −80°C for subsequent biochemical analysis. 
 
3.7 Serum Preparation and Storage 
Approximately 10 mL of whole blood from all study participants will be 
collected in a dry tube. The tubes will then be kept for one hour at 37°C to allow 
coagulum retraction. Next, they will be centrifuged at 2,000 rpm for 10 minutes, 
and the serum will be separated, aliquoted into microtubes, and stored in an ultra-
freezer at –80°C. 
 
3.8 Isolation of Extracellular Vesicles (EVs) 
The isolation of EVs will follow the mitochondrial separation procedure 
described by Higuchi et al29. Serum samples will be diluted (1:5) in a dense, 
sugar-rich medium containing 200 mM D-mannitol, 70 mM sucrose, 2 mM 
HEPES, and 0.5 g/L BSA (pH 7.2), and incubated at room temperature for one 
hour. Samples will then be centrifuged for 12 minutes at 9,500 g, and the 
supernatants will be collected for further processing. 
 
 
 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
13 
 
3.9 Morphological Analysis of EVs by Transmission Electron 
Microscopy (TEM) 
Morphological evidence of EVs will be obtained using Transmission 
Electron Microscopy (TEM). Sample embedding will follow the rapid procedure 
described by Duarte et al30. etect exosomes and microvesicles present in the 
supernatant after phase separation, 400 μL of the supernatant will be fixed by 
adding 1 mL of 3% glutaraldehyde at 4°C for 3 hours, followed by postfixation 
with a reduced osmium solution (700 μL of 1% osmium tetroxide + 700 μL of 3% 
potassium ferricyanide) at 4°C for 30 minutes, and centrifugation at 13,000 g for 
5 minutes at 12°C. This procedure allows pellet formation, as lipid-rich exosomes 
and microvesicles become heavier after fixation. The pellet will then be collected 
and washed in a solution containing 0.9% NaCl + 360 mOsm/kg sucrose, and 
incubated in 0.5% uranyl acetate for 3 hours at 4°C. 
The pellets will then be dehydrated for 10 minutes in 70% ethanol, 5 minutes 
in acidified 2,2-dimethoxypropane with 0.1 N HCl, and 2 minutes in 4% copper 
sulfate in acetone. Infiltration will be performed using a mixture of EponEMbed 
812 resin and araldite 502 resin (1:1), and polymerization will take place in an 
oven at 100°C for 1 hour. Blocks will be sectioned using an ultramicrotome at 60–
70 nm thickness and placed on palladium-coated copper–nickel grids (200 mesh) 
(Electron Microscopy Sciences, Hatfield, PA, USA). 
 
 
 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
14 
 
3.10 Protein Signature of EVs (LC/MS/MS) 
The proteomic profiling of serum EVs will be performed using liquid 
chromatography coupled with mass spectrometry (LC/MS). After isolation, the 
supernatant containing EVs will undergo protein extraction with RIPA buffer (150 
mM NaCl, 50 mM Tris pH 8, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) 
containing protease inhibitors without EDTA. Protein concentration will be 
determined via spectrometry using the Bradford method31., according to a 
standard BSA calibration curve. 
An aliquot corresponding to 100 μg of total protein from each sample will be 
incubated with sample buffer containing ¼ β-mercaptoethanol, and heated at 
100°C for 5 minutes for protein denaturation. Samples will then be subjected to 
10% polyacrylamide gel electrophoresis at 140 V for 1 hour. After electrophoresis, 
gels will be rinsed with deionized water, proteins will be fixed, and the gel will be 
stained for 1 hour using Coomassie Blue R-350. Protein bands will be excised, 
recovered, and digested with trypsin following the protocol described by Lundby 
et a32. Extracted peptides will be analyzed on a high-resolution LC/MS system, 
allowing identification of proteins of interest. 
 
3.11 Quantification and Characterization by Nanosight – NTA 
(Nanoparticle Tracking Analysis) 
A Serum EV characterization (size, concentration, and protein signature) 
will be performed using a highly sensitive nanoparticle tracking method 
(Nanosight – NTA). Using a NanoSight NS300 ultramicroscope equipped with a 
high-sensitivity sCMOS camera (Malvern Instruments, Ltd., Malvern, UK), 
particle detection will be carried out by light scattering when irradiated by a laser 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
15 
 
beam, and particle size will be determined according to the Stokes–Einstein 
equation, based on Brownian motion. 
Serum-derived EVs will be diluted (1:50) in particle-free PBS (0.02 µm 
filtered) to achieve a concentration within the recommended measurement range 
(1–10×10⁸ particles/mL). Immunolabeling of predefined proteins will follow 
antibody datasheet recommendations, and detection will be performed using the 
appropriate fluorophore–laser wavelength combination. 
Image acquisition settings will include: automatic camera level, manual 
threshold and focus adjustment, 25°C measurement temperature, 5 runs at 30 
fps, and 60-second recordings. Scattered light images will be automatically 
analyzed using NTA software (version 3.4). Light-scattering dot plots and 
movement-velocity graphs will then be generated, enabling quantification of 
unlabeled and/or immunolabeled particles (Malvern Panalytical, Ltd., Malvern, 
UK). 
 
3.12 Statistical Analysis 
Statistical analyses will be performed using GraphPad Prism 6. Each 
variable will first be assessed for normality using the Kolmogorov–Smirnov 
distance test, and classified as parametric or non-parametric. 
Parametric (normally distributed) data will be presented as mean ± 
standard deviation, and group comparisons will be performed by ANOVA, 
followed by Tukey’s post-hoc test. Non-parametric (non-normal) data will be 
presented as median and interquartile range, and comparisons will be made 
using the Kruskal–Wallis test, followed by Dunn’s post-hoc test. 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
16 
 
Spearman’s correlation test will be performed to assess potential 
associations between EV characteristics and the degree of myocardial 
dysfunction across groups, as well as imaging findings. A p-value < 0.05 will be 
considered statistically significant.   
   
3.13 Ethical Considerations 
 The principal investigator will ensure that Informed Consent (Appendix 1) 
is obtained in accordance with international ethical and regulatory guidelines 
established by the ICH (International Council for Harmonisation) for Good Clinical 
Practice and the ethical principles defined by the Brazilian National Health 
Council Resolution No. 466/2012. 
This study will be submitted for approval by the Research Ethics Committee 
(CAPPesq) via Plataforma Brasil. All ethical considerations involving human 
research defined by Resolution 466/2012 will be followed. 
All collected data will be treated confidentially and in compliance with the 
Brazilian General Data Protection Law (LGPD). After study completion, data will 
be anonymized and analyzed by the InCor research team. Upon final analysis, 
results are expected to be published in scientific journals.  
 
3.14 Risk and Benefit Assessment of the Clinical Protocol 
3.14.1 Risks 
During participation, some procedures may cause mild discomfort or 
minimal risks. ECG may cause mild skin adhesion sensation or, rarely, allergic 
reactions to electrode adhesives. Echocardiography is painless, although the 
conductive gel may cause cold or wet sensations, with rare gel-related allergic 
reactions. Technetium pyrophosphate scintigraphy involves slight discomfort 
during injection and minimal radiation exposure, with very low risk of allergic 
reactions to the radiopharmaceutical. MRI may be uncomfortable for individuals 
with claustrophobia, and gadolinium contrast carries risks for patients with renal 
impairment. Blood collection may cause needle puncture discomfort, bruising, 
and post-collection dizziness. 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
17 
 
3.14.2 Benefits 
Expected benefits include comprehensive cardiac health evaluation 
through advanced imaging and diagnostic tools that enable early detection of 
abnormalities related to cardiac amyloidosis and other conditions. 
Participation also contributes to the advancement of scientific knowledge 
on cardiac amyloidosis and its early diagnosis. 
Investigating extracellular vesicles as potential biomarkers may lead to 
more sensitive and specific diagnostic methods and more targeted and effective 
therapies, benefiting not only participants but also the broader medical 
community and future patients. 
 
 
1 Gilstrap LG, Dominici F, Wang Y, El-Sady MS, Singh A, Di Carli MF, et al. Epidemiology 
of Cardiac Amyloidosis-Associated Heart Failure Hospitalizations Among Fee-for-
Service Medicare Beneficiaries in the United States. Circ Heart Fail. 
2019;12(6):e005407. 
 
2 Donnelly, J. P., & Hanna, M. (2017). Cardiac amyloidosis: An update on diagnosis and 
treatment. Cleveland Clinic journal of medicine, 84(12 Suppl 3), 12-26. 
 
3 Alexander, KM, Orav, J., Singh, A., Jacob, SA, Menon, A., Padera, RF, ... e Dorbala, S. 
(2018). Disparidades geográficas na mortalidade por amiloidose nos Estados Unidos 
relatada de 1979 a 2015: potencial subdetecção de amiloidose cardíaca. JAMA 
cardiology , 3 (9), 865-870. 
4 Banypersad, S. M., Moon, J. C., Whelan, C., Hawkins, P. N., & Wechalekar, A. D. 
(2012). Updates in cardiac amyloidosis: a review. Journal of the American Heart 
Association, 1(2), e000364. 
 
5 Maleszewski, J. J. (2015). Cardiac amyloidosis: pathology, nomenclature, and typing. 
Cardiovascular Pathology, 24(6), 343-350. 
 
6 Damy, T., Costes, B., Hagège, A. A., Donal, E., Eicher, J. C., Slama, M., ... & Buxbaum, J. 
N. (2016). Prevalence and clinical phenotype of hereditary transthyretin amyloid 
cardiomyopathy in patients with increased left ventricular wall thickness. European 
heart journal, 37(23), 1826-1834. 
 
7 Mankad, A. K., & Shah, K. B. (2017). Transthyretin cardiac amyloidosis. Current 
cardiology reports, 19(10), 1-11. 
 
8 Ruberg, F. L., & Berk, J. L. (2012). Transthyretin (TTR) cardiac amyloidosis. Circulation, 
126(10), 1286-1300. 
 
 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
18 
 
 
9 Galat, A., Guellich, A., Bodez, D., Slama, M., Dijos, M., Zeitoun, D. M., ... & Damy, T. 
(2016). Aortic stenosis and transthyretin cardiac amyloidosis: the chicken or the egg?. 
European heart journal, 37(47), 3525-3531. 
 
10 Treibel, T. A., Fontana, M., Gilbertson, J. A., Castelletti, S., White, S. K., Scully, P. R., ... 
& Moon, J. C. (2016). Occult transthyretin cardiac amyloid in severe calcific aortic 
stenosis: prevalence and prognosis in patients undergoing surgical aortic valve 
replacement. Circulation: Cardiovascular Imaging, 9(8), e005066. 
 
11 González-López, E., Gagliardi, C., Dominguez, F., Quarta, C. C., de Haro-Del Moral, F. 
J., Milandri, A., ... & Garcia-Pavia, P. (2017). Clinical characteristics of wild-type 
transthyretin cardiac amyloidosis: disproving myths. European heart journal, 38(24), 
1895-1904. 
 
12 Maurer, M. S., Elliott, P., Comenzo, R., Semigran, M., & Rapezzi, C. (2017). Addressing 
common questions encountered in the diagnosis and management of cardiac 
amyloidosis. Circulation, 135(14), 1357-1377. 
 
13 Fontana, M., Pica, S., Reant, P., Abdel-Gadir, A., Treibel, T. A., Banypersad, S. M., ... & 
Moon, J. C. (2015). Prognostic value of late gadolinium enhancement cardiovascular 
magnetic resonance in cardiac amyloidosis. Circulation, 132(16), 1570-1579. 
 
14 Gillmore, J. D., Maurer, M. S., Falk, R. H., Merlini, G., Damy, T., Dispenzieri, A., ... & 
Hawkins, P. N. (2016). Nonbiopsy diagnosis of cardiac transthyretin amyloidosis. 
Circulation, 133(24), 2404-2412. 
 
15 Stremersch, S., De Smedt, SC, & Raemdonck, K. (2016). Aplicações terapêuticas e 
diagnósticas de vesículas extracelulares. Journal of Controlled Release , 244 , 167-183. 
 
16 Roy, S., Hochberg, FH, & Jones, PS (2018). Vesículas extracelulares: o crescimento 
como diagnóstico e terapêutica; um questionário. Journal of extracellular vesicles , 7 
(1), 1438720. 
 
17 Sunkara, V., Woo, HK e Cho, YK (2016). Técnicas emergentes no isolamento e 
caracterização de vesículas extracelulares e seus papéis no diagnóstico e prognóstico 
do câncer. Analista , 141 (2), 371-381. 
 
18 Ridger VC, Boulanger CM, Angelillo-Scherrer A, Badimon L, Blanc-Brude O, Bochaton- 
Piallat ML, Boilard E, et al. Microvesicles in vascular homeostasis and diseases. Position 
Paper of the European Society of Cardiology (ESC) Working Group on Atherosclerosis 
and Vascular Biology. ThrombHaemost. 2017; 117(7):1296-1316. 
 
19 Barry OP, FitzGerald GA. Mechanisms of cellular activation by platelet 
microparticles.ThrombHaemost. 1999; 82:794–800 
 
 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
19 
 
 
20 Van Der Pol, E., Hoekstra, A. G., Sturk, A., Otto, C., Van Leeuwen, T. G., & Nieuwland, 
R. (2010). Optical and non‐optical methods for detection and characterization of 
microparticles and exosomes. Journal of Thrombosis and Haemostasis, 8(12), 2596-
2607. 
 
21 Bakhshandeh, B., Amin Kamaleddin, M., & Aalishah, K. (2017). A comprehensive 
review on exosomes and microvesicles as epigenetic factors. Current stem cell 
research & therapy, 12(1), 31-36. 
 
22 Roura, S., Gámez-Valero, A., Lupón, J., Gálvez-Montón, C., Borràs, F. E., & Bayes-
Genis, A. (2018). Proteomic signature of circulating extracellular vesicles in dilated 
cardiomyopathy. Laboratory Investigation, 98(10), 1291-1299. 
 
23 Waldenström, A., & Ronquist, G. (2014). Role of exosomes in myocardial remodeling. 
Circulation research, 114(2), 315-324. 
 
24 Ramirez‐Alvarado, M., Barnidge, D. R., Murray, D. L., Dispenzieri, A., Marin‐Argany, 
M., Dick, C. J., ... & Leung, N. (2017). Assessment of renal response with urinary 
exosomes in patients with AL amyloidosis: A proof of concept. American journal of 
hematology, 92(6), 536-541. 
 
25 Ramirez-Alvarado, M., Ward, C. J., Huang, B. Q., Gong, X., Hogan, M. C., Madden, B. 
J., ... & Leung, N. (2012). Differences in immunoglobulin light chain species found in 
urinary exosomes in light chain amyloidosis (Al). PLoS One, 7(6), e38061. 
 
26 Pérez‐González, R., Kim, Y., Miller, C., Pacheco‐Quinto, J., Eckman, E. A., & Levy, E. 
(2020). Extracellular vesicles: where the amyloid precursor protein carboxyl‐terminal 
fragments accumulate and amyloid‐β oligomerizes. The FASEB Journal, 34(9), 12922-
12931. 
 
27 Santucci, L., Bruschi, M., Del Zotto, G., Antonini, F., Ghiggeri, G. M., Panfoli, I., & 
Candiano, G. (2019). Biological surface properties in extracellular vesicles and their 
effect on cargo proteins. Scientific reports, 9(1), 1-12. 
 
28 Keller, M.D., Ching, K.L., Liang F.X.,Dhabaria, A., Tam,K., Ueberheide B.M.,…& 
Cadwell, K. (2020). Decoy exosomes provide protection against bacterial toxins. 
Nature, 579 (7798) 260-264. 
 
29 Higuchi, M. D. L., Kawakami, J. T., Ikegami, R. N., Reis, M. M., Pereira, J. D. J., Ianni, B. 
M., ... & Bocchi, E. A. (2018). Archaea symbiont of T. cruzi infection may explain heart 
failure in Chagas disease. Frontiers in cellular and infection microbiology, 8, 412. 
 
30 Duarte MIS, Mariano ON, Takakura CFH. A fast method for processing biologic 
material for electron-microscopic diagnosis in infectious-disease. Ultrastructural 
Pathology. [Article]. 1992 Jul-Aug;16(4):475-82 
 
 

 
 
 
Versão 1.0 de 10/01/24               
Projeto InCor 
20 
 
 
31 Bradford MM.A rapid and sensitive method for the quantitation of microgram 
quantities of protein utilizing the principle of protein-dye binding.AnalBiochem. 1976; 
72:248-54. 
 
32 Lundby, A., & Olsen, J. V. (2011). GeLCMS for in-depth protein characterization and 
advanced analysis of proteomes. In Gel-Free Proteomics (pp. 143-155). Humana Press.