Periodic Reporting for period 2 - MACROVALVE (Understanding the role of macrophages in the structural degeneration of bioprosthetic heart valve leaflets)
Período documentado: 2024-09-01 hasta 2025-08-31
The prevalence of aortic valve (AV) stenosis in individuals aged 75 years or older is 12.4%, and the incidence increases with age. Accordingly, the number of patients requiring valve replacement annually is expected to 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 two 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 investigate the contribution of host macrophages to leaflet degeneration under biomechanical loading.
The overall goal of the fellowship is to uncover processes underlying bioprosthetic (BP) heart valve degeneration. This goal is broken down into two 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 investigate the contribution of host macrophages to leaflet degeneration under biomechanical loading.
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 by Prof Aikawa’s group, 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 proteomics lab at CICS (Thermo Lumos and Exploris Orbitrap instruments). The fellow has gained expertise in proteomic sample preparation, mass spectrometry sequencing, and proteomic data analysis.
The fellow developed a proteomics pipeline suitable for the processing of cross-species xenogenic cardiovascular biomaterials. Created a proteomic map comparing segmented degenerated bioprosthetic and diseased native aortic valve tissue, suggesting novel subtypes of bioprosthetic degeneration and calcification. Histopathology confirmed four distinct regions of bioprosthetic degeneration (non-degenerated, plasma insudation, surface thrombosis and neotissue), each capable of calcifying. Laser capture microdissection enabled spatial proteomics of the distinct regions, demonstrating that the proteins involved in the calcific remodelling of each region are largely unique. The fellow identified, for the first time, extracellular vesicles in explanted, degenerated bioprosthetic valve tissue in comparison with non-implanted control and compared their cargos with extracellular vesicles from native aortic valves. This is the first comparative multidimensional proteomic study of degenerated BP valves and AV disease.
The incoming phase of the fellowship was conducted at the Mechanobiology and Medical Device Research Group at the University of Galway. The group led by Prof McNamara is recognised for their expertise in mechanobiology and developing cellular mechanobiology assays. Multiphysics simulations from the McNamara group were used to determine the solid and fluid stress ranges for the experiments. The fellow developed a setup to study inflammatory cells under shear flow and the tensile properties of glutaraldehyde-fixed bovine pericardium. Conditioned media from cells under shear affected the tensile properties of bovine pericardium.
The fellow developed a proteomics pipeline suitable for the processing of cross-species xenogenic cardiovascular biomaterials. Created a proteomic map comparing segmented degenerated bioprosthetic and diseased native aortic valve tissue, suggesting novel subtypes of bioprosthetic degeneration and calcification. Histopathology confirmed four distinct regions of bioprosthetic degeneration (non-degenerated, plasma insudation, surface thrombosis and neotissue), each capable of calcifying. Laser capture microdissection enabled spatial proteomics of the distinct regions, demonstrating that the proteins involved in the calcific remodelling of each region are largely unique. The fellow identified, for the first time, extracellular vesicles in explanted, degenerated bioprosthetic valve tissue in comparison with non-implanted control and compared their cargos with extracellular vesicles from native aortic valves. This is the first comparative multidimensional proteomic study of degenerated BP valves and AV disease.
The incoming phase of the fellowship was conducted at the Mechanobiology and Medical Device Research Group at the University of Galway. The group led by Prof McNamara is recognised for their expertise in mechanobiology and developing cellular mechanobiology assays. Multiphysics simulations from the McNamara group were used to determine the solid and fluid stress ranges for the experiments. The fellow developed a setup to study inflammatory cells under shear flow and the tensile properties of glutaraldehyde-fixed bovine pericardium. Conditioned media from cells under shear affected the tensile properties of bovine pericardium.
Glutaraldehyde-fixed bovine pericardium valves are the predominantly used devices in the clinic. 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 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). 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 as building blocks of cardiovascular calcification, an ultrastructural assessment of explanted degenerated bioprosthetic tissue was conducted, confirming the presence of EVs. Using tissue EV isolation strategies developed at CICS, EVs were isolated from degeneration BP tissue for downstream processing and proteomic analysis.
The pathogenesis of BHV degeneration likely stems from complex interactions between the biomechanical environment, host response, and leaflet matrix. During each cardiac cycle, pressure changes cause the aortic valve to open and close, inducing repeated flexural deformation, radial stresses, and shear forces on the leaflets. Although the solid biomechanics of valve tissue have been widely studied to predict fatigue and tearing, recent clinical and explanted tissue studies have observed that shear stress correlates with the incidence of degeneration and calcification. The fellow observed the acquisition of circulating inflammatory cells at the BHV blood-contacting surface. However, the role of inflammatory cells in BHV degeneration, especially in response to shear stress, remains poorly understood. Experiments were designed to determine whether differential shear patterns experienced by acquired host inflammatory cells at the BHV blood contacting surface drive bioprosthetic degeneration.
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 as building blocks of cardiovascular calcification, an ultrastructural assessment of explanted degenerated bioprosthetic tissue was conducted, confirming the presence of EVs. Using tissue EV isolation strategies developed at CICS, EVs were isolated from degeneration BP tissue for downstream processing and proteomic analysis.
The pathogenesis of BHV degeneration likely stems from complex interactions between the biomechanical environment, host response, and leaflet matrix. During each cardiac cycle, pressure changes cause the aortic valve to open and close, inducing repeated flexural deformation, radial stresses, and shear forces on the leaflets. Although the solid biomechanics of valve tissue have been widely studied to predict fatigue and tearing, recent clinical and explanted tissue studies have observed that shear stress correlates with the incidence of degeneration and calcification. The fellow observed the acquisition of circulating inflammatory cells at the BHV blood-contacting surface. However, the role of inflammatory cells in BHV degeneration, especially in response to shear stress, remains poorly understood. Experiments were designed to determine whether differential shear patterns experienced by acquired host inflammatory cells at the BHV blood contacting surface drive bioprosthetic degeneration.