Periodic Reporting for period 1 - MACROVALVE (Understanding the role of macrophages in the structural degeneration of bioprosthetic heart valve leaflets)
Periodo di rendicontazione: 2022-09-01 al 2024-08-31
The prevalence of aortic valve (AV) stenosis in those ≥75 years of age is 12.4% and the incidence increases with age. Accordingly, the number of patients requiring valve replacement yearly will rise to over 850,000 globally by 2050. Currently, the only treatment option for heart valve disease is valve replacement. Valves can be sutured into place during open-heart surgery (surgical) or crimped onto a catheter and delivered percutaneously (transcatheter). Structural valve degeneration (SVD) is the inevitable and irreversible remodelling of a bioprosthetic valve which limits the device lifespan, up to one-third of patients exhibit subclinical SVD after only 8 years. Additionally, specific devices and patient populations are at risk of accelerated SVD. However, the mechanisms underlying SVD are still not fully understood.
The overall goal of the fellowship is to uncover processes underlying bioprosthetic (BP) heart valve degeneration. This goal is broken down into 2 objectives: 1) to examine the regulatory molecular networks and 2) to elucidate the role of host inflammatory macrophages. Mass spectrometry has become the leading modality for analysing complex protein samples. Therefore objective 1 set out to conduct a histopathological assessment of BP degeneration and build proteomic comparison maps of BP degeneration versus native AV disease. Within this cohort, subanalyses will be conducted where possible to determine the effect of patient factors such as sex, age, and length of implantation on subsequent bioprosthetic degeneration. Secondly, the immune response is a critical determinant of implant longevity. Therefore, the second objective proposes to resolve the contribution of host macrophages to leaflet degeneration.
The overall goal of the fellowship is to uncover processes underlying bioprosthetic (BP) heart valve degeneration. This goal is broken down into 2 objectives: 1) to examine the regulatory molecular networks and 2) to elucidate the role of host inflammatory macrophages. Mass spectrometry has become the leading modality for analysing complex protein samples. Therefore objective 1 set out to conduct a histopathological assessment of BP degeneration and build proteomic comparison maps of BP degeneration versus native AV disease. Within this cohort, subanalyses will be conducted where possible to determine the effect of patient factors such as sex, age, and length of implantation on subsequent bioprosthetic degeneration. Secondly, the immune response is a critical determinant of implant longevity. Therefore, the second objective proposes to resolve the contribution of host macrophages to leaflet degeneration.
The outgoing phase of this fellowship was conducted at the Center for Interdisciplinary Cardiovascular Sciences (CICS) at Brigham and Women’s Hospital, Harvard Medical School. Following the well-established disease segmentation procedure for native valves at CICS, the fellow developed a novel degeneration subtype segmentation procedure for degenerated bioprosthetic valves. Using the state-of-the-art histopathology facilities at CICS, the fellow received individualised histopathology training to examine the degenerated bioprosthetic samples. Segmented bioprosthetic tissues were analysed using mass spectrometry-based proteomics, facilitated by the state-of-the-art proteomics lab at CICS (Thermo Lumos and Exploris instruments). The fellow received training in proteomic sample preparation, protein extraction, digestion, and purification for mass spectrometry injection from proteomics experts. Following this, the fellow’s expertise in proteomics was evident by being selected as an abstract finalist in the basic science category at the Heart Valve Society meeting 2024 and being included as an expert on the supplementary proteomics workshop.
The fellow received training in the field of extracellular vesicles (EVs) by taking the International Society of Extracellular Vesicles (ISEV) open online courses (Basics of Extracellular Vesicles, Extracellular Vesicles in Health and Disease, and Detection and Isolation of Intact Extracellular Vesicles) and following the ISEV annual education day videos on YouTube. Bioprosthetic tissue segments were processed and sent to the Electron Microscopy Core at Harvard Medical School to determine if EVs were present. Training in sample preparation and the use of a transmission electron microscope and ultrastructural analysis was provided. Experimental training in isolating and characterising tissue-entrapped EVs using protocols previously developed at CICS was received through research. Tissue-entrapped EVs were isolated from the bioprosthetic valves by enzymatic digestion, serial ultracentrifugation, and density-gradient separation. The proteomic profile of acquired tissue-entrapped EVs was acquired.
Inflammatory cell research was initiated by collaborating with Dr Masanori Aikawa’s research group on macrophage biology and inflammation. Peripheral blood mononuclear cells were isolated from the blood samples of bioprosthetic valve patients at Brigham and Women’s Hospital. THP-1 cell culture training was provided. Initial experiments were performed by the fellow and her research student. THP-1 cell interaction with non-implanted bioprosthetic material was investigated.
The fellow received training in the field of extracellular vesicles (EVs) by taking the International Society of Extracellular Vesicles (ISEV) open online courses (Basics of Extracellular Vesicles, Extracellular Vesicles in Health and Disease, and Detection and Isolation of Intact Extracellular Vesicles) and following the ISEV annual education day videos on YouTube. Bioprosthetic tissue segments were processed and sent to the Electron Microscopy Core at Harvard Medical School to determine if EVs were present. Training in sample preparation and the use of a transmission electron microscope and ultrastructural analysis was provided. Experimental training in isolating and characterising tissue-entrapped EVs using protocols previously developed at CICS was received through research. Tissue-entrapped EVs were isolated from the bioprosthetic valves by enzymatic digestion, serial ultracentrifugation, and density-gradient separation. The proteomic profile of acquired tissue-entrapped EVs was acquired.
Inflammatory cell research was initiated by collaborating with Dr Masanori Aikawa’s research group on macrophage biology and inflammation. Peripheral blood mononuclear cells were isolated from the blood samples of bioprosthetic valve patients at Brigham and Women’s Hospital. THP-1 cell culture training was provided. Initial experiments were performed by the fellow and her research student. THP-1 cell interaction with non-implanted bioprosthetic material was investigated.
Native calcific aortic valve disease and bioprosthetic structural valve degeneration share risk factors and have end-stage features in common. While the mechanisms of BP degeneration are not fully known, there are likely multiple processes involved, with certain pathophysiological mechanisms, such as inflammation and calcification, that could be shared with native aortic valve disease. Therefore, to date, the fellow has conducted a histopathological assessment of BP degeneration and built proteomic comparison maps of BP degeneration versus AV disease in the human aortic position across three tissue length scales.
Glutaraldehyde-fixed bovine pericardium valves are the predominantly used devices. Proteomic analysis of explanted degeneration bioprosthetics is, therefore, a cross-species challenge, as the degenerated explants are suspected to contain proteins from both the donor species (Bos taurus) and the host species (Homo sapiens). However, in traditional bottom-up proteomic approaches, shared peptide amino acid (AA) sequences across donor versus host species render protein-level specificity a challenge. Previous bioprosthetic research has been limited by the lack of an appropriate processing workflow to consider the background proteome of the xenogenic ECM (glutaraldehyde-fixed bovine pericardium or porcine aortic valves). During this fellowship, for the first time, a proteomic pipeline was developed including non-implanted BP tissues to perform species differentiation and exclude any non-implanted BP background proteins.
Previous bioprosthetic (BP) research has been limited by the disregard for the heterogeneous nature of degeneration. Guided by gross pathological features and a review of the histopathology literature, we defined for the first time subtypes of BP degeneration and then conducted label-free proteomic profiling, in comparison with well-established disease stage-associated regions of native aortic valve disease. In the histopathological analyses, different regions of calcification were identified in BP. The fellow used laser capture microdissection to microscopically isolate these different subtypes of bioprosthetic calcification for independent downstream processing and proteomic analysis. Finally, as extracellular vesicles (EVs) have been implicated in the pathogenesis of aortic stenosis and are widely appreciated building blocks of cardiovascular calcification, an ultrastructural assessment of explanted degenerated bioprosthetic tissue was conducted and confirmed the presence of EVs. Using tissue EV isolation strategies developed at CICS, EVs were from degeneration BP tissue for downstream processing and proteomic analysis.
Glutaraldehyde-fixed bovine pericardium valves are the predominantly used devices. Proteomic analysis of explanted degeneration bioprosthetics is, therefore, a cross-species challenge, as the degenerated explants are suspected to contain proteins from both the donor species (Bos taurus) and the host species (Homo sapiens). However, in traditional bottom-up proteomic approaches, shared peptide amino acid (AA) sequences across donor versus host species render protein-level specificity a challenge. Previous bioprosthetic research has been limited by the lack of an appropriate processing workflow to consider the background proteome of the xenogenic ECM (glutaraldehyde-fixed bovine pericardium or porcine aortic valves). During this fellowship, for the first time, a proteomic pipeline was developed including non-implanted BP tissues to perform species differentiation and exclude any non-implanted BP background proteins.
Previous bioprosthetic (BP) research has been limited by the disregard for the heterogeneous nature of degeneration. Guided by gross pathological features and a review of the histopathology literature, we defined for the first time subtypes of BP degeneration and then conducted label-free proteomic profiling, in comparison with well-established disease stage-associated regions of native aortic valve disease. In the histopathological analyses, different regions of calcification were identified in BP. The fellow used laser capture microdissection to microscopically isolate these different subtypes of bioprosthetic calcification for independent downstream processing and proteomic analysis. Finally, as extracellular vesicles (EVs) have been implicated in the pathogenesis of aortic stenosis and are widely appreciated building blocks of cardiovascular calcification, an ultrastructural assessment of explanted degenerated bioprosthetic tissue was conducted and confirmed the presence of EVs. Using tissue EV isolation strategies developed at CICS, EVs were from degeneration BP tissue for downstream processing and proteomic analysis.