TTR Deposition Research Articles

Vitronectin promotes TTR deposition and mitochondrial damage in ATTR-CA mice

Abstract Background Transthyretin cardiac amyloidosis (ATTR-CA) is an increasingly recognized disorder that leads to heart failure and cardiovascular death. However, the pathogenesis of ATTR-CA is still poorly understood. Vitronectin (VTN), a common component of the extracellular matrix, has been reported to be associate with amyloidosis. However, the role of VTN in TTR deposition has not been clarified. Purpose Here we aimed to elucidate the TTR deposition mechanism in ATTR-CA and test therapeutic interventions using a gene-edited ATTR-CA mice model. Methods We used CRISPR/Cas9-mediated genome engineering to create humanized wild-type TTR knock-in mice (hTTRWT). Cardiac function was examined by echocardiography and pressure-volume analysis. VTN expression was assessed by immunofluorescence and western blotting. Interactions among VTN and TTR are analyzed with in vitro pull-down and co-immunoprecipitation (Co-IP) assays. Recombinant TTR protein was added in the culture of VTN overexpressed mice primary cardiomyocytes (CMs) to simulate the TTR deposition process in vitro. Mitochondrial function was determined by the Seahorse assay and mitochondrial ultrastructure was evaluated with the transmission electron microscope.Additionally, we assessed the mitochondrial damage with mitoSOX staining. Results In the hTTRWT group, pressure-volume analysis and echocardiography revealed significant diastolic dysfunction and increased left ventricle chamber stiffness at 52 weeks. Compared to WT group, the hTTRWT group showed a dramatic elevation of VTN in hearts. Co-IP and pull-down assays confirmed a direct interaction between VTN and TTR. In cultured CMs exposed to recombinant TTR protein, VTN overexpression promotes the deposition of TTR. Damaged mitochondrial ultrastructure, elevated oxidative stress and decreased mitochondrial numbers were found. In accordance with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity. Conclusion These results suggest that the VTN contributes to ATTR-related pathological and cardiac function changes and mitochondrial dysfunction as a contributor of disease progression from ATTR-CA to heart failure.

P5 DISCOVERY OF A NEW HATTR GENETIC VARIANT: A CASE REPORT OF A PATIENT WITH CARDIAC UPTAKE AT 99–MTC–DIPHOSPHONATES SCINTIGRAPHY AND ABNORMAL MONOCLONAL PROTEINS TESTS

Abstract Introduction Cardiac amyloidosis is an infiltrative disease that remains an under–diagnosticated cause of heart failure. Diagnostic algorithms focus on identifying the two most frequent forms of the disease, light chains amyloidosis (AL) and transthyretin amyloidosis (ATTR), by the initial use of 99mTc–DPD scintigraphy coupled to assessment of monoclonal proteins. Etiological distinction is and of pivotal importance since the availability of specific disease modifying treatments. Case Description We report the case of a 61–year–old man hospitalized for new onset of heart failure. Past medical history highlights bilateral carpal tunnel syndrome and monoclonal gammopathy of undetermined significance (MGUS). Cardiovascular examination reveals clinical signs of pulmonary and peripheric congestion. ECG shows conduction disturbances and low QRS voltage. Transthoracic echocardiography reveals left ventricular hypertrophy, normal systolic function (LVEF 58%), 3rd degree LV diastolic disfunction and mild pericardial effusion. Laboratory tests shows abnormal levels of TnT and pro–BNP. A diagnosis of cardiac amyloidosis is considered, and further exams are executed: cardiac magnetic resonance, haematological laboratory tests and 99mTc–DPD scintigraphy (tab. 1). Since both bone scintigraphy and monoclonal proteins tests are positive, diagnosis of cardiac amyloidosis cannot be made without non–invasive modalities (fig.1) and the patient is referred to an Amyloidosis Centre. Haematological evaluation and abdominal fat pad biopsy show stability of haematological disease and confirms the deposition of amyloidogenic TTR both in wild–type and mutated Ile88Arg variants. The results of genetic analysis confirms the presence of a heterozygous mutation of TTR gene (p.Ile88Arg – c.263T>G) (fig. 2). This mutation is not reported in database ClinVar nor in gnomAD, but according to the ACMG criteria it can be considered likely pathogenic. Considering the final diagnosis of cardiac hATTR the patient is considered eligible for treatment with Tafamidis. Discussion and Conclusion Despite clinical history with cardiac conduction system abnormalities and bilateral carpal tunnel syndrome, some gene TTR variants can bring false negative results at 99mTc–DPD scintigraphy, so ATTR form should not be excluded basing on non–invasive modalities. Performing a compete diagnostic workup led us to discover a new TTR pathogenic variant and confirm ATTR form without performing endomyocardial biopsy.

Cellular environment of TTR deposits in an animal model of ATTR-Cardiomyopathy.

Introduction: Cardiac amyloidoses are the most fatal manifestation of systemic amyloidoses. It is believed the number of cases to be greatly underestimated mostly due to misdiagnosis. Particularly, the involvement of TTR V30M in the heart of ATTRV30M amyloidosis has not been completely understood specifically in terms of implicated cellular pathways, heart function and cardiac physiology. In the present work we proposed to characterize TTR V30M cardiac involvement particularly at the tissue cellular level in a mouse model. Methods: HSF ± hTTR V30M mice, a model that expresses human TTRV30M in a Ttr null background, widely used for the characterization and modulation of neurological features of ATTRV30M amyloidosis was used. SDS-PAGE of cardiac homogenates followed by Western blot was performed. Immunohistochemistry and double immunofluorescence analyses were carried out to determine TTR deposition pattern and sub-localization. Results: Western blots were able to detect TTR in its monomeric state at ∼14kDa. Immunofluorescent images showed TTR was found mostly in the intercellular spaces. Blood contamination was excluded by CD31 staining. Tissues were Congo Red negative. Upon TTR and macrophages (CD68) staining in the cardiac tissue a clear tendency of macrophage convergence to the tissue regions where TTR was more abundant was observed. Moreover, in some instances it was possible to detect co-localization of both fluorophores. Cardiac fibroblasts were stained with PDGFr-alpha, and here the co-localization was not so evident although there was some degree of co-occurrence. The hearts of transgenic mice revealed higher content of Galectin-3. Conclusion: This animal model and associated features observed as result of cardiac TTR deposition provide a promising and invaluable research tool for a better understanding of the implicated pathways that lead to the lethality associated to TTR cardiac amyloidosis. New therapeutic strategies can be tested and ultimately this will lead to improved treatment alternatives capable of increasing patient's quality of life and life expectancy and, hopefully to eradicate a condition that is silently spreading worldwide.

Inotersen to Treat Polyneuropathy Associated with Hereditary Transthyretin (hATTR) Amyloidosis.

Amyloidosis is a group of diseases with the common pathophysiology of protein misfolding and aberrant deposition in tissue. There are both acquired and hereditary forms of this disease, and this review focuses on the latter hereditary transthyretin-mediated (hATTR). hATTR affects about 50,000 individuals globally and mostly appears as one of three syndromes - cardiac, polyneuropathy, and oculoleptomeningeal. Polyneuropathy is the most common form, and there is usually some overlap in individual patients. Recently, novel therapeutic options emerged in the form of groundbreaking drugs, Patisiran and Inotersen, small interfering RNA molecules that target TTR and reduce the production of this protein. By targeting TTR mRNA transcripts, Inotersen decreases protein translation and production, reducing the deposition of misfolded proteins. It was shown to be both effective and safe for use and specifically formulated to concentrate in the liver - where protein production takes place. hATTR is a rare, progressive, and debilitating disease. Its most common presentation is that of polyneuropathy, and it carries a very poor prognosis and a natural history conveying a median survival of < 12 years. Novel therapeutic options are groundbreaking by providing disease-modifying specific, targeted therapies against TTR production and deposition. The use of RNA interference (RNAi) opens the door to the treatment of hereditary diseases by targeting them at the genetic level.

Liver-directed drugs for transthyretin-mediated amyloidosis.

Transthyretin-mediated amyloidosis (ATTR) is a rare, under-recognized, progressively debilitating, fatal disease caused by the aggregation and extracellular deposition of amyloid transthyretin (TTR) fibrils in multiple organs and tissues throughout the body. TTR is predominantly synthesized by the liver and normally circulates as a homotetramer, while misfolded monomers aggregate to form amyloid fibrils. One strategy to treat ATTR amyloidosis is to reduce the amount of TTR produced by the liver using drugs that directly target the TTR mRNA or gene. This narrative review focuses on how TTR gene silencing tools act to reduce TTR production, describing strategies for improved targeted delivery of these agents to hepatocytes where TTR is preferentially expressed. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), termed RNA silencers, cause selective degradation of TTR mRNA, while a TTR gene editing tool reduces TTR expression by introducing nonsense mutations into the TTR gene. Two strategies to facilitate tissue-specific delivery of these nucleic acid-based drugs employ endogenous receptors expressed by hepatocytes. Lipid nanoparticles (LNPs) that recruit apolipoprotein E support low-density lipoprotein receptor-mediated uptake of unconjugated siRNA and are now used for CRISPR gene editing tools. Additionally, conjugating N-acetylgalactosamine (GalNAc) moieties to ASOs or siRNAs facilitates receptor-mediated uptake by the asialoglycoprotein receptor. In summary, ATTR is a progressive disease with various clinical manifestations due to TTR aggregation, deposition, and amyloid formation. Receptor-targeted ligands (eg, GalNAc) and nanoparticle encapsulation (eg, LNPs) are technologies to deliver ASOs, siRNAs, and gene editing tools to hepatocytes, the primary location of TTR synthesis.

Abstract 15440: Elevated Serum Transthyretin Concentration Found to be Associated With Increased Survival Rate in Hereditary Transthyretin Cardiomyopathy Patients

Introduction: Transthyretin amyloid cardiomyopathy (ATTR-CM) results from abnormal deposition of misfolded transthyretin (TTR) deposition in the myocardium. Prior studies have shown an association between lower serum concentrations of TTR with increased mortality in patients with wild-type TTR, suggesting that greater deposition of TTR from the blood into surrounding tissues may reflect increased disease severity. Hypothesis: Serum TTR levels are associated with mortality and HF hospitalization in hATTR patients with Val142Ile mutation. Methods: We conducted a retrospective chart review of patients diagnosed with hATTR-CM at Emory University's Cardiac Amyloidosis Clinic between January 2014 and April 2022 (n=65) and evaluated the association of serum TTR concentration with 1 year survival and 1 year HF hospitalization. We stratified patients on two TTR cut-offs: 18 mg/dL, based on a previous study in a wild-type cohort, and 14.9 mg/dL, the median TTR level in this cohort. Kaplan Meier Survival analysis and Cox proportional hazard modeling examined the association between serum TTR, overall survival, and HF hospitalization, adjusted for troponin-I, age, BMI, BNP, GFR and NYHA class as covariates. Results: Increased serum concentrations of TTR were significantly associated with decreased mortality within 1 year of diagnosis in multivariable models. Adjusted models showed a significant difference between the groups at both cut-offs only when the event was death within 1 year of diagnosis (Cutoff 14.9: HR = 14.02, 95% CI 2.4 - 82.5; cutoff 18: HR 367, 95% CI 35.24-3822.57), but no significant difference for the outcome of HF hospitalization at either cutoff point ( Table ). Conclusions: In our cohort of 65 hATTR patients with the Val142Ile mutation, lower TTR levels at the time of diagnosis were significantly associated with death at 1 year of diagnosis. TTR levels were not significantly associated with HF hospitalization within 1 year of diagnosis.

Multifunctional Therapeutic Cyclodextrin-Appended Dendrimer Complex for Treatment of Systemic and Localized Amyloidosis.

Amyloidosis pathologically proceeds via production of amyloidogenic proteins by organs, formation of protein aggregates through structural changes, and their deposition on tissues. A growing body of evidence demonstrates that amyloidosis generally develops through three critical pathological steps: (1) production of amyloid precursor proteins, (2) amyloid formation, and (3) amyloid deposition. However, no clinically effective therapy that is capable of targeting each pathological step of amyloidosis independently is currently available. Here, we combined therapeutic effects and developed a short hairpin RNA expression vector (shRNA) complex with a cyclodextrin-appended cationic dendrimer (CDE) as a novel multitarget therapeutic drug that is capable of simultaneously suppressing these three steps. We evaluated its therapeutic effects on systemic transthyretin (ATTR) amyloidosis and Alzheimer's disease (AD) as localized amyloidosis, by targeting TTR and amyloid β, respectively. CDE/shRNA exhibited RNAi effects to suppress amyloid protein production and also achieved both inhibition of amyloid formation and disruption of existing amyloid fibrils. The multitarget therapeutic effects of CDE/shRNA were confirmed by evaluating TTR deposition reduction in early- and late-onset human ATTR amyloidosis model rats and amyloid β deposition reduction in AppNL-G-F/NL-G-F AD model mice. Thus, the CDE/shRNA complex exhibits multifunctional therapeutic efficacy and may reveal novel strategies for establishing curative treatments for both systemic and localized amyloidosis.

Transthyretin Amyloid Cardiomyopathy: Impact of Transthyretin Amyloid Deposition in Myocardium on Cardiac Morphology and Function.

Background: Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is increasingly being recognized as a cause of left ventricular (LV) hypertrophy (LVH) and progressive heart failure in elderly patients. However, little is known about the cardiac morphology of ATTR-CM and the association between the degree of TTR amyloid deposition and cardiac dysfunction in these patients. Methods: We studied 28 consecutive patients with ATTR-CM and analyzed the relationship between echocardiographic parameters and pathological features using endomyocardial biopsy samples. Results: The cardiac geometries of patients with ATTR-CM were mainly classified as concentric LVH (96.4%). The relative wall thickness, a marker of LVH, tended to be positively correlated with the degree of non-cardiomyocyte area. The extent of TTR deposition was positively correlated with enlargement of the non-cardiomyocyte area, and these were positively correlated with LV diastolic dysfunction. Additionally, the extent of the area containing TTR was positively correlated with the percentage of cardiomyocyte nuclei stained for 8-hydroxy-2′deoxyguanosine, a marker of reactive oxygen species (ROS). ROS accumulation in cardiomyocytes was positively correlated with LV systolic dysfunction. Conclusion: Patients with ATTR-CM mainly displayed concentric LVH geometry. TTR amyloid deposition was associated with cardiac dysfunction via increased non-cardiomyocyte area and ROS accumulation in cardiomyocytes.

High-avidity binding drives nucleation of amyloidogenic transthyretin monomer.

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursors. Transthyretin (TTR) naturally folds into a stable tetramer, whereas conditions and mutations that foster aberrant monomer formations facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the early assembly of oligomers by WT TTR compared with its V30M and V122I variants. We monitored time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20–40 TTR subunits via engineered biotin-streptavidin (SA) interactions. As expected, these multimer seeds rapidly nucleated TTR monomers into larger complexes, while having less effect on dimers and tetramers. In vivo, SA-induced multimers formed TTR-like deposits in the heart and the kidney following i.v. injection in mice. While all 3 variants prominently deposited glomerulus in the kidney, only V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized in the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR engaging in high-avidity interactions with tissue amyloids. Our multimeric induction approach provides a model for studying the initiation of TTR deposition in the heart.

Incidence And Demographics Of Transthyretin Amyloidosis-Positive Subjects Identified By Histologic Analysis Of Tenosynovial Tissue Obtained During Carpal Tunnel Surgery

<h3>Background</h3> Transthyretin (TTR) cardiac amyloidosis (CA) is an increasingly recognized cause of heart failure (HF) with preserved ejection fraction (HFpEF) that is associated with a poor prognosis. Retrospective analyses reported that amyloidogenic TTR (ATTR) deposition in tenosynovial sheaths such as that causing carpal tunnel syndrome often precedes the onset of HF due to ATTR CA by several years. <h3>Methods</h3> We conducted an analysis of 135 consecutive subjects who presented for carpel tunnel surgery from which tenosynovial sheaths were analyzed for evidence of amyloid deposition to explore the frequency of samples positive for ATTR and whether this approach might lead to earlier diagnosis and treatment of ATTR CA. <h3>Results</h3> From these 135 subjects, 31 were confirmed to have positive Congo Red staining deposits within tenosynovial tissue obtained during carpal tunnel release, all of which demonstrated ATTR subtype determined by liquid chromatography/mass-spectroscopic analysis. ATTR positive patients were significantly older than the negative patients (74 ± 8 yrs vs. 59±13 yrs). 55<i>%</i> of the ATTR positive patients were male and 45% female. At the time of this submission, only 1 of these 31 demonstrated a TTR mutation (valine-isoleucine 122). Seventeen patients have undergone screening for cardiac involvement of which 2 men have been diagnosed with ATTR CA by bone scintigraphic imaging. <h3>Conclusion</h3> 23% of subjects undergoing carpal tunnel release exhibit amyloid deposition by histologic analysis, all of whom tested positive for the ATTR subtype, with 2 male subjects demonstrating cardiac involvement. Within our positive ATTR population, 45% are female which far exceeds the 20% percent female prevalence among patients with HF due to ATTR CA reported previously supporting that female gender may offer protection from the subsequent development of ATTR CA. The evaluation of this population is ongoing and will be reported at the annual meeting. Broadly, our data support that histologic testing for amyloid protein deposition within tenosynovial tissue obtained at the time of surgery is warranted as this approach likely facilitates early diagnosis and treatment of ATTR CA before the onset of overt clinical heart failure.

Abstract 9749: High-Avidity Binding Drives Nucleation of Amyloidogenic Transthyretin Monomer in Cardiac Amyloidosis

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursor proteins. Mutations of plasma carrier protein transthyretin (TTR) are associated with hereditary transthyretin amyloidosis (ATTR). TTR naturally folds into a stable tetramer for its function, whereas conditions and mutations that foster aberrant monomer formation facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the dynamics of early assembly of oligomers by wild-type TTR, in comparison to its TTR V30M and TTR V122I disease mutants. We selected buffer conditions for monitoring time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20-40 TTR subunits via engineered biotin-streptavidin interactions. As expected, these multimer seeds rapidly nucleated TTR monomers and dimers into larger complexes, while having no effect on tetramers. In vivo , streptavidin-induced multimers formed ATTR-like deposits in the heart and the kidney following i.v. injection in mice. While all three variants prominently deposited in the kidney glomerulus, only TTR V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized to the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR to engage high-avidity interactions with tissue amyloids. Our multimeric induction approach therefore provides a new model for studying the initiation of TTR deposition in the heart.

Abstract 11707: Incidence and Clinical Features of Transthyretin Amyloidosis Diagnosed by Histologic Analysis of Tenosynovial Tissue From Patients Undergoing Carpal Tunnel Surgery

Introduction: Transthyretin (TTR) cardiac amyloidosis (CA) is an increasingly recognized cause of heart failure (HF) with preserved ejection fraction (HFpEF) that is associated with a poor prognosis. Retrospective series have demonstrated that amyloidogenic TTR (ATTR) deposition in tenosynovial sheaths such as that causing carpal tunnel syndrome precedes the onset of HF due to ATTR CA by several years. Methods: We conducted an analysis of 136 patients (pts) who presented for carpel tunnel surgery from whom tenosynovial material was analyzed with Congo Red staining to determine the frequency of samples positive for ATTR deposition and to explore whether this approach might lead to earlier diagnosis and treatment of ATTR CA. Results: Among these 136 pts, 31 were confirmed to have positive Congo Red staining tenosynovial tissue, all of which demonstrated the ATTR subtype determined by liquid chromatography/mass-spectroscopic analysis. ATTR positive pts were significantly older than negative pts (74 + 8 yrs vs. 59 + 13 yrs, p&lt;0.01). 58 % of ATTR positive pts were male and 42% female. At the time of this submission, only 1 of these 31 subjects demonstrated a TTR mutation (valine-isoleucine 122). 19 pts have undergone cardiac screening of which 2 males demonstrated ATTR CA by bone scintigraphic imaging. Conclusions: 23% of pts undergoing carpal tunnel release exhibit ATTR amyloid deposition by histologic analysis, all of whom tested positive for the ATTR subtype with 2 asymptomatic male pts exhibiting cardiac involvement. Among our positive ATTR pts, 42% are female exceeding the 10-20% percent female prevalence among patients with HF due to ATTR CA reported previously. This preliminary observation supports a protective role of female gender in limiting progression to HF due to ATTR amyloidosis. The evaluation of this population is ongoing and will be reported at the annual meeting. Broadly, however, our data support that identifying ATTR amyloid protein within tenosynovial tissue obtained during routine surgery is warranted as this approach, when combined with appropriate cardiac screening, facilitates early diagnosis and treatment of ATTR CA before the onset of overt clinical heart failure.

Surveillance for disease progression of transthyretin amyloidosis after heart transplantation in the era of novel disease modifying therapies

Heart Transplantation (HT) is a rational therapy for advanced transthyretin cardiac amyloidosis (ATTR-CA), but the impact of ongoing amyloid deposition is not well defined. We evaluated a cohort of patients who underwent HT for ATTR-CA to determine the incidence of de novo or progression of post-HT ATTR deposition. All patients who were followed post-HT for ATTR-CA at our center were included. Baseline demographics and post-HT manifestations of TTR deposition were collected. All patients completed the Composite Autonomic Symptom Score (COMPASS-31 quantifies autonomic symptoms, with a higher score [0-100] indicating more severe autonomic dysfunction) and Polyneuropathy Disability Score (PND, range from 0 [asymptomatic] to IV [confined to wheelchair/bed]) questionnaires. Twelve patients (5 wild-type, 7 variant [6 p.Val142Ile, 1 p.Thr80Ala]) were included. Mean age at HT was 64.6 (SD: 4.8) years, 83.3% male, and 50% Black. At a median of 4.0 years (IQR 2.4, 5.9) post-HT, 8 patients had symptoms of ATTR deposition (5 with gastrointestinal involvement, 4 orthopedic and 4 neurologic), with 4 patients having ≥2 body systems involved. There were no patients with recurrent cardiac involvement. Median COMPASS-31 score was 17.3 (IQR 11.3, 23.5) at 3.9 years (IQR 2.4, 5.9) post-HT. Four patients had a PND score of stage 1 (sensory disturbance), 1 patient was stage 2 (impaired walking) and 1 patient stage 3b (required a walking aid). More than 50% of patients had evidence of progressive or de novo ATTR deposition post-HT, impairing quality of life despite a well-functioning cardiac allograft. These observations highlight an unmet need to establish the role of formal surveillance and treatment of TTR using TTR disease-modifying therapies, which may maintain or improve quality of life post-HT for ATTR-CA.

Concomitant wild type transthyretin amyloidosis &amp; severe aortic stenosis in elderly Indian population: pilot study

Abstract Background Prevalence of both severe aortic stenosis (AS) and wild-type transthyretin cardiac amyloidosis (wtTTRCA) increases with age. Dual disease (AS+wtTTRCA) occurs in a significant proportion of patients undergoing aortic valve replacement. Prognostic significance of this dual disease is not established. Purpose TTRCA distribution has ethnic variability. This has not been studied in an Indian/Asian population before. India alone has &amp;gt;4.6 million estimated elderly individuals with severe AS. This pilot study was designed as part of a larger study to determine the prevalence of coexistence of severe AS and wtTTRCA in elderly Indian population and identify non-invasive predictors of its diagnosis. Methods Symptomatic severe AS patients aged ≥65 years undergoing surgical aortic valve replacement (SAVR) were enrolled. Detailed 2-D transthoracic echocardiogram including speckle tracking and longitudinal strain assessment was done preoperatively. TTRCA diagnosis was based on preoperative 99m-Technitium pyrophosphate scan (PYP), and intra-operatively obtained basal inter-ventricular septum (IVS) biopsy and excised native aortic valve subjected to histopathological examination (HPE). Congo red staining and immunohistochemistry using TTR monospecific antibodies were performed to identify and sub-type amyloid respectively. Serum/urine protein electrophoresis with serum immunofixation to rule out primary amyloidosis and genetic analysis to rule out mutant TTRCA were also done. Results SAVR was done in 46 severe AS patients (mean age 70±5 years, 70% men), 32 (70%) of whom also underwent PYP scan. Significant PYP uptake (Perrugini grade II or III) was seen in 3 (9%) patients (Figure 1E, F). On HPE, 33 (72%) native aortic valves had amyloid deposits, of which 19 (58%) had TTR deposition (Figure 1A, B, C, D). However, none of the IVS biopsies showed amyloid deposits. None of the patients had evidence of monoclonal protein elevation and mutation in the TTR gene. Non-invasive markers of this dual coexistence included elevated troponin-I (≥26.2 pg/mL, 35% vs 67%), combined elevated troponin-I &amp; NT-ProBNP (≥26.2 pg/mL &amp; ≥400 pg/mL, 24% vs 33%), and significantly low myocardial contraction fraction (≤30%, 72% vs 100%), deceleration time (207±80 msec vs 85±23 msec) and global longitudinal strain (−19.3±4.3% vs −13.6±3.7%). At median follow-up of 12.5 months, 1 patient each had died in TTR negative and TTR positive groups (3% vs 33%). The results are summarised in the central illustration (Figure 2). Conclusion In the Indian context, concomitant existence of severe AS and wtTTRCA is common. This pilot study suggests an absolute burden of &amp;gt;450,000 patients with dual disease of AS and wtTTRCA, in those otherwise believed to have lone severe AS. Further larger studies are thus indicated to establish the prevalence of this dual disease and to assess its prognostic implication. Funding Acknowledgement Type of funding sources: None. Figure 1. Histopathology and PYP scan resultsFigure 2. Central illustration

In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis.

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.

Concurrent cardiac transthyretin and brain β amyloid accumulation among the older adults: The Hisayama study.

Previous studies have revealed risk for cognitive impairment in cardiovascular diseases. We investigated the relationship between degenerative changes of the brain and heart, with reference to Alzheimer's disease (AD) pathologies, cardiac transthyretin amyloid (ATTR) deposition, and cardiac fibrosis. A total of 240 consecutive autopsy cases of a Japanese population‐based study were examined. β amyloid (Aβ) of senile plaques, phosphorylated tau protein of neurofibrillary tangles, and ATTR in the hearts were immunohistochemically detected and graded according to the NIH‐AA guideline for AD pathology and as Tanskanen reported, respectively. Cerebral amyloid angiopathy (CAA) was graded according to the Vonsattel scale. Cardiac fibrosis was detected by picrosirius red staining, followed by image analysis. Cardiac ATTR deposition occurred after age 75 years and increased in an age‐dependent manner. ATTR deposition was more common, and of higher grades, in the dementia cases. We subdivided the cases into two age groups: ≤90 years old (n = 173) and >90 years old (n = 67), which was the mean and median age at death of the AD cases. When adjusted for age and sex, TTR deposition grades correlated with Aβ phase score (A2–3), the Consortium to Establish a Registry for AD score (sparse to frequent), and high Braak stage (V–VI) only in those aged ≤90 years at death. No significant correlation was observed between the cardiac ATTR deposition and CAA stages, or between cardiac fibrosis and AD pathologies. Collectively, AD brain pathology correlated with cardiac TTR deposition among the older adults ≤90 years.

Surveillance for Transthyretin Amyloidosis Progression after Heart Transplantation in the Era of Novel Disease Modifying Therapies

<h3>Purpose</h3> Transthyretin amyloidosis (ATTR) predominantly involves the heart (ATTR-cardiac amyloidosis [CA]) and nerves, and may involve other organs or tissues. Disease modifying therapies improve outcomes in those with ATTR-CA or with neurologic involvement, however their utility post heart transplant (HT) or combined heart/liver transplant is uncertain. We evaluated patients who underwent HT for ATTR-CA to determine the incidence of de novo development or progression of neurologic, GI and soft tissue manifestations of ATTR deposition. <h3>Methods</h3> We performed a review of all patients who underwent HT for ATTR-CA at our center. Baseline demographics and post HT manifestations of TTR deposition were collected. All patients completed the Composite Autonomic Symptom Score (COMPASS-31) and Polyneuropathy Disability Score (PDS). The COMPASS-31 is a questionnaire of autonomic symptoms, with a higher score (0 - 100) indicating more severe autonomic dysfunction. The PDS ranges from 0 (asymptomatic) to 4 (confined to wheelchair/bed). <h3>Results</h3> Twelve patients were included. Mean age at HT was 67 ± 5 yrs, 83.3% male, and 50% Black. Five patients had wild-type and 7 had variant ATTR-CA (6 V122I, 1 T60A). At a mean of 5.2 ± 3.9 yrs post-transplant, 8 patients had symptoms of progressive or de novo ATTR deposition with 4 having involvement in ≥ 2 systems (Figure 1). None had cardiac involvement on PYP imaging (n=5) or serial endomyocardial biopsy. The mean COMPASS-31 was 19.5 (range 0.9 - 45.8) at a median time from HT of 3.9 ± 3.5 years. Three subjects had a PDS of 1 and 1 each scored 2, 3b, and 4, the latter patient was homozygous for the V122I variant. <h3>Conclusion</h3> In our single-center study, > 50% of patients had evidence of progressive or de novo TTR deposition post HT. This adds significant morbidity despite a functioning cardiac allograft. These observations underscore an unmet need for TTR modifying therapies that could slow TTR deposition and significantly improve quality of life post HT for ATTR-CA.

Drug Discovery and Development in Rare Diseases: Taking a Closer Look at the Tafamidis Story.

Rare diseases are increasingly recognized as a global public health priority. Governments worldwide currently provide important incentives to stimulate the discovery and development of orphan drugs for the treatment of these conditions, but substantial scientific, clinical, and regulatory challenges remain. Tafamidis is a first-in-class, disease-modifying transthyretin (TTR) kinetic stabilizer that represents a major breakthrough in the treatment of transthyretin amyloidosis (ATTR amyloidosis). ATTR amyloidosis is a rare, progressive, and fatal systemic disorder caused by aggregation of misfolded TTR and extracellular deposition of amyloid fibrils in various tissues and organs, including the heart and nervous systems. In this review, we present the successful development of tafamidis spanning 3 decades, marked by meticulous laboratory research into disease mechanisms and natural history, and innovative clinical study design and implementation. These efforts established the safety and efficacy profile of tafamidis, leading to its regulatory approval, and enabled post-approval initiatives that further support patients with ATTR amyloidosis.

Transthyretin Amyloidosis: Update on the Clinical Spectrum, Pathogenesis, and Disease-Modifying Therapies.

ATTR amyloidosis is caused by systemic deposition of transthyretin (TTR) and comprises ATTRwt (wt for wild-type) amyloidosis, ATTRv (v for variant) amyloidosis, and acquired ATTR amyloidosis after domino liver transplantation. ATTRwt amyloidosis has classically been regarded as cardiomyopathy found in the elderly, whereas carpal tunnel syndrome has also become a major initial manifestation. The phenotypes of ATTRv amyloidosis are diverse and include neuropathy, cardiomyopathy, and oculoleptomeningeal involvement as the predominant features, depending on the mutation and age of onset. In addition to variant TTR, the deposition of wild-type TTR plays a significant role, even in patients with ATTRv amyloidosis. The formation of amyloid fibrils tends to occur in association with the basement membrane. The thickening or reduplication of the basement membrane surrounding endoneurial microvessels, which is similar to diabetic neuropathy, is observed in ATTRv amyloidosis, suggesting that common mechanisms, such as an accumulation of advanced glycation end products, may participate in the disease process. In addition to direct damage caused by amyloid fibrils, recent studies have suggested that the toxicity of nonfibrillar TTRs, such as TTR oligomers, participates in the process of tissue damage. Although liver transplantation has been performed for patients with ATTRv amyloidosis since 1990, late-onset patients were not eligible for this treatment. However, as the efficacy of orally administered tafamidis and diflunisal, which stabilize TTR tetramers, was suggested in the early 2010s, such late-onset patients have also become targets for disease-modifying therapies. Additionally, recent studies of small interfering RNA (patisiran) and antisense oligonucleotide (inotersen) therapies have demonstrated the efficacy of these gene-silencing agents. A strategy for monitoring patients that enables the choice of an appropriate treatment from comprehensive and long-term viewpoints should be established. As many patients with ATTR amyloidosis are aged and have heart failure, they are at increased risk of aggravation if they are infected by SARS-CoV2. The optimal interval of evaluation should also be considered, particularly in this COVID-19 era.

SERPINA1 modulates expression of amyloidogenic transthyretin

Hereditary transthyretin amyloidosis (ATTR) is caused by amyloid deposition of misfolded transthyretin (TTR) in various tissues. Recently, reduction of circulating serum TTR, achieved via silencing oligonucleotides, was introduced as therapy of ATTR amyloidosis. We explored the impact of Serpin Family A Member 1 (SERPINA1) on TTR mRNA and protein expression. Oncostatin M (OSM) induced SERPINA1 in hepatoma cells and mice, while concomitantly TTR expression was significantly reduced. SERPINA1 knockdown resulted in specific elevated TTR expression in hepatoma cells; however other genes belonging to the group of acute phase proteins were unaffected. In mice, serum TTR was elevated after mSERPINA1 knockdown throughout antisense treatment. Following SERPINA1 knockdown, TTR deposition in several tissues, including dorsal root ganglia and intestine, was found to be increased, however numbers did not exceed significance levels. The data suggest that SERPINA1 is a co-factor of TTR expression. Our findings provide novel insight in the regulation of TTR and reveal a role of SERPINA1 in the pathogenesis of ATTR amyloidosis.