Extracellular Matrix Biology Research Articles

Integrated proteomics and metabolomics analyses reveal new insights into the antitumor effects of valproic acid plus simvastatin combination in a prostate cancer xenograft model associated with downmodulation of YAP/TAZ signaling.

Despite advancements in therapeutic approaches, including taxane-based chemotherapy and androgen receptor-targeting agents, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable tumor, highlighting the need for novel strategies that can target the complexities of this disease and bypass the development of drug resistance mechanisms. We previously demonstrated the synergistic antitumor interaction of valproic acid (VPA), an antiepileptic agent with histone deacetylase inhibitory activity, with the lipid-lowering drug simvastatin (SIM). This combination sensitizes mCRPC cells to docetaxel treatment both in vitro and in vivo by targeting the cancer stem cell compartment via mevalonate pathway/YAP axis modulation. Here, using a combined proteomic and metabolomic/lipidomic approach, we characterized tumor samples derived from 22Rv1 mCRPC cell-xenografted mice treated with or without VPA/SIM and performed an in-depth bioinformatics analysis. We confirmed the specific impact of VPA/SIM on the Hippo-YAP signaling pathway, which is functionally related to the modulation of cancer-related extracellular matrix biology and metabolic reprogramming, providing further insights into the molecular mechanism of the antitumor effects of VPA/SIM. In this study, we present an in-depth exploration of the potential to repurpose two generic, safe drugs for mCRPC treatment, valproic acid (VPA) and simvastatin (SIM), which already show antitumor efficacy in combination, primarily affecting the cancer stem cell compartment via MVP/YAP axis modulation. Bioinformatics analysis of the LC‒MS/MS and 1H‒NMR metabolomics/lipidomics results confirmed the specific impact of VPA/SIM on Hippo-YAP.

Editorial comments to “Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology” (JPUROL-D-24-00006)

Editorial comments to “Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology” (JPUROL-D-24-00006)

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro.

After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-β1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-β1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

Editorial commentary to “Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology”

Editorial commentary to “Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology”

Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology

Acellular matrices have historically been applied as biologic scaffolds in surgery, wound care, and tissue engineering, albeit with inconsistent outcomes. One aspect that varies widely between products is the selection of decellularization protocol, yet few studies assess comparative effectiveness of these protocols in preserving mechanics, and protein content. This study characterizes bladder acellular matrix (BAM) using two different detergent and enzymatic protocols, evaluating effects on nuclei and DNA removal (≥90%), structure, tensile properties, and maintenance of extracellular matrix proteins. Porcine bladders were decellularized with 0.5% Sodium Dodecyl Sulfate (SDS) or 0.25% Trypsin-hypotonic-Triton X-100 hypertonic (TT)-based agitation protocols, followed by DNase/RNase agents. Characterization of BAM included decellularization efficacy (DAPI, DNA quantification), structure (histology and scanning electron microscopy), tensile testing (Instron 345C-1 mechanical tester), and protein presence and diversity (mass spectrometry). SDS and TT data was directly compared to the same native bladder using two-tailed paired t-tests. Native, TT, and SDS cohorts for tensile testing were compared using one-way ANOVA; Tukey's post-hoc tests for among group differences. Effective nuclei removal was achieved by SDS- and TT-based protocols. However, target DNA removal was achieved with SDS but not TT. SDS more effectively maintained qualitative tissue architecture compared to TT. The tensile modulus of the TT cohort increased, and stretchability decreased after decellularization in both SDS and TT. UTS was unaffected by either protocol. Higher overall diversity and quantity of core matrisome and matrisome-associated proteins was maintained in the SDS vs TT cohort post-decellularization. The results indicated that detergent selection affects multiple aspects of the resultant BAM biologic product. In the selected protocols, SDS was superior to TT efficacy, and maintenance of gross tissue architecture as well as maintenance of ECM proteins. Decellularization increased scaffold resistance to deformation in both cohorts. Future studies applying biologic scaffolds must consider the processing method and agents used to ensure that materials selected are optimized for characteristics that will facilitate effective translational use.

Characterizing the immune response to myocardial infarction in pigs

Though myocardial infarction (MI) in pigs is a well-established translational large animal model, it has not yet been widely used for immunotherapy studies, and a comprehensive description of the immune response to MI in this species is lacking. We induced MI in Landrace pigs by balloon occlusion of the left anterior descending artery over 90 min. Within 14 days, the necrotic myocardium was progressively replaced by scar tissue with involvement of myofibroblasts. We characterized the immune response in the heart ex vivo by (immuno)histology, flow cytometry, and RNA sequencing of myocardial tissue on days 3, 7, and 14 after MI. Besides a clear predominance of myeloid cells among heart-infiltrating leukocytes, we detected activated T cells and an increasing proportion of CD4+ Foxp3+ regulatory T cells (Treg), especially in the infarct core—findings that closely mirror what has been observed in mice and humans after MI. Transcriptome data indicated inflammatory activity that was persistent but markedly changing in character over time and linked to extracellular matrix biology. Analysis of lymphocytes in heart-draining lymph nodes revealed significantly higher proliferation rates of T helper cell subsets, including Treg on day 7 after MI, compared to sham controls. Elevated frequencies of myeloid progenitors in the spleen suggest that it might be a site of emergency myelopoiesis after MI in pigs, as previously shown in mice. We thus provide a first description of the immune response to MI in pigs, and our results can aid future research using the species for preclinical immunotherapy studies.

Extracellular matrix in synovium development, homeostasis and arthritis disease

Extracellular matrix (ECM) is a three-dimensional network entity composed of extracellular macromolecules. ECM in synovium not only supports the structural integrity of synovium, but also plays a crucial role in regulating homeostasis and damage repair response in synovium. Obvious disorders in the composition, behavior and function of synovial ECM will lead to the occurrence and development of arthritis diseases such as rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis (PsA). Based on the importance of synovial ECM, targeted regulation of the composition and structure of ECM is considered to be an effective measure for the treatment of arthritis disease. This paper reviews the current research status of synovial ECM biology, discusses the role and mechanism of synovial ECM in physiological status and arthritis disease, and summarizes the current strategies for targeting synovial ECM to provide information for the pathogenesis, diagnosis and treatment of arthritis disease.

Genome-wide association meta-analysis of spontaneous coronary artery dissection identifies risk variants and genes related to artery integrity and tissue-mediated coagulation

Spontaneous coronary artery dissection (SCAD) is an understudied cause of myocardial infarction primarily affecting women. It is not known to what extent SCAD is genetically distinct from other cardiovascular diseases, including atherosclerotic coronary artery disease (CAD). Here we present a genome-wide association meta-analysis (1,917 cases and 9,292 controls) identifying 16 risk loci for SCAD. Integrative functional annotations prioritized genes that are likely to be regulated in vascular smooth muscle cells and artery fibroblasts and implicated in extracellular matrix biology. One locus containing the tissue factor gene F3, which is involved in blood coagulation cascade initiation, appears to be specific for SCAD risk. Several associated variants have diametrically opposite associations with CAD, suggesting that shared biological processes contribute to both diseases, but through different mechanisms. We also infer a causal role for high blood pressure in SCAD. Our findings provide novel pathophysiological insights involving arterial integrity and tissue-mediated coagulation in SCAD and set the stage for future specific therapeutics and preventions.

A roadmap for developing and engineering in vitro pulmonary fibrosis models.

Idiopathic pulmonary fibrosis (IPF) is a severe form of pulmonary fibrosis. IPF is a fatal disease with no cure and is challenging to diagnose. Unfortunately, due to the elusive etiology of IPF and a late diagnosis, there are no cures for IPF. Two FDA-approved drugs for IPF, nintedanib and pirfenidone, slow the progression of the disease, yet fail to cure or reverse it. Furthermore, most animal models have been unable to completely recapitulate the physiology of human IPF, resulting in the failure of many drug candidates in preclinical studies. In the last few decades, the development of new IPF drugs focused on changes at the cellular level, as it was believed that the cells were the main players in IPF development and progression. However, recent studies have shed light on the critical role of the extracellular matrix (ECM) in IPF development, where the ECM communicates with cells and initiates a positive feedback loop to promote fibrotic processes. Stemming from this shift in the understanding of fibrosis, there is a need to develop in vitro model systems that mimic the human lung microenvironment to better understand how biochemical and biomechanical cues drive fibrotic processes in IPF. However, current in vitro cell culture platforms, which may include substrates with different stiffness or natural hydrogels, have shortcomings in recapitulating the complexity of fibrosis. This review aims to draw a roadmap for developing advanced in vitro pulmonary fibrosis models, which can be leveraged to understand better different mechanisms involved in IPF and develop drug candidates with improved efficacy. We begin with a brief overview defining pulmonary fibrosis and highlight the importance of ECM components in the disease progression. We focus on fibroblasts and myofibroblasts in the context of ECM biology and fibrotic processes, as most conventional advanced in vitro models of pulmonary fibrosis use these cell types. We transition to discussing the parameters of the 3D microenvironment that are relevant in pulmonary fibrosis progression. Finally, the review ends by summarizing the state of the art in the field and future directions.

An Impact of Hypoxia and Macromolecular Crowders on Extracellular Matrix Deposition by Human Endometrial Mesenchymal Stromal Cells

The last decades are characterized by intensive development of extracellular matrix (ECM) biology. ECM binds cells in an integral tissue and controls the cell functions – from proliferation and differentiation to migration and apoptosis. Bioactive properties of ECM provide the wide perspectives of using in bioengineering and regenerative medicine. In this context, the ECM production by decellularization of organs, tissues or cell cultures is a key technology. To date, a problem of a rapid and large-scale production of bioactive ECM by cultured cells remains very relevant. Optimization of the ECM deposition conditions by human endometrial mesenchymal stromal cells (MESCs) had not been studied yet. Here, we investigated an impact of macromolecular compounds (crowders) – ficoll and PEG on efficiency of crucial ECM proteins deposition depending on both concentration and molecular weight of crowders under normoxia and hypoxia. According to immunofluorescence analysis, among all studied crowders, ficoll 400 had a potent effect on the production of ECM core proteins – fibronectin, type IV collagen and, in a lower rate, type III collagen. The MESCs incubation under hypoxia promoted the formation of a properly organized ECM structure as well as increase in efficiency of ECM protein deposition. Of note, in these conditions ficoll 400 accelerated the ECM production only in а low serum medium. Together, combination of ficoll 400, low serum medium and hypoxia provides the optimal conditions for ECM synthesis. The present work demonstrates for the first time the phenomenon of macromolecular crowding in the context of improving the conditions for deposition and organization of ECM by MESCs.

A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI‐NET sequencing

Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non‐coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA‐seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA‐seq, we delineated a deep intronic TAPT1 mutation (c.1237‐52 G>A) that segregated with the disease. Using SI‐NET‐seq, we document that TAPT1's nascent transcription was not affected in patients' fibroblasts, indicating instead that this variant leads to an alteration of pre‐mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation enhances TAPT1 exon 12 skipping, creating a protein‐null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease‐relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussions of non‐coding variants, as well as in illuminating the molecular mechanisms of human diseases.

Understanding conceptus–maternal interactions: what tools do we need to develop?

Communication between the maternal endometrium and developing embryo/conceptus is critical to support successful pregnancy to term. Studying the peri-implantation period of pregnancy is critical as this is when most pregnancy loss occurs in cattle. Our current understanding of these interactions is limited, due to the lack of appropriate in vitro models to assess these interactions. The endometrium is a complex and heterogeneous tissue that is regulated in a transcriptional and translational manner throughout the oestrous cycle. While there are in vitro models to study endometrial function, they are static and 2D in nature or explant models and are limited in how well they recapitulate the in vivo endometrium. Recent developments in organoid systems, microfluidic approaches, extracellular matrix biology, and in silico approaches provide a new opportunity to develop in vitro systems that better model the in vivo scenario. This will allow us to investigate in a more high-throughput manner the fundamental molecular interactions that are required for successful pregnancy in cattle.

Detergent-Free Decellularization Preserves the Mechanical and Biological Integrity of Murine Tendon.

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. In this study, we adapted and established the advantages of a detergent-free decellularization method that relies on latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Our method maintained the collagen molecular structure (i.e., minimal extent of denaturation), while adequately removing cells and preserving bulk mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/"provisional"), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact statement In this study, we present a decellularization method for tendon that does not rely on any detergent or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of gross fiber architecture, collagen molecular structure, and extracellular matrix (ECM)-associated biological cues that are essential for studying physiological cell-ECM interactions. Finally, we demonstrated the applicability of this method for healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

Degradomics technologies in matrisome exploration

Consisting of a defined set of extracellular proteins secreted from resident cells and with minor contributions from serum proteins, the extracellular matrix (ECM) is an essential component of all tissues. Maintaining tissue homeostasis, structural support and cellular control through cell-ECM communication, the ECM has come to be viewed as not just a passive structural entity but rather as a dynamic signaling conduit between cells and the extracellular compartment. Proteins and their cleavage products mediate this communication, and aberrant signaling, either directly or indirectly distorting the ECM, results in pathological conditions including cancer, inflammation, fibrosis, and neurodegenerative diseases. Characterization of ECM components, the matrisome, the extracellular environment and their changes in disease is therefore of importance to understand and mitigate by developing novel therapeutics. Liquid chromatography-mass spectrometry (LC-MS) proteomics has been integral to protein and proteome research for decades and long superseded the obsolescent gel-based approaches. A continuous effort has ensured progress with increased sensitivity and throughput as more advanced equipment has been developed hand in hand with specialized enrichment, detection, and identification methods. Part of this effort lies in the field of degradomics, a branch of proteomics focused on discovering novel protease substrates by identification of protease-generated neo-N termini, the N-terminome, and characterizing the responsible protease networks. Various methods to do so have been developed, some specialized for specific tissue types, others for particular proteases, throughput, or ease of use. This review aims to provide an overview of the state-of-the-art proteomics techniques that have successfully been recently utilized to characterize proteolytic cleavages in the ECM and thereby guided new research and understanding of the ECM and matrisome biology.

80 (PB070) - Human EGFR (HER) family ligands as targets in Cancer and Autoimmune Disease

Background: Solid tumors and rheumatoid arthritis have overlapping mechanisms of pathogenesis. Both diseases are characterized by a hyperproliferative component, hypoxia, excessive angiogenesis, and inflammation. First-line treatments also overlap. Examples are methotrexate and cyclophosphamide which are used to treat many different malignancies as well as being first-line treatments for rheumatoid arthritis. The hyperproliferative component of RA is the pannus, which is a fibroblastic mass that expands in the affected joint and provides a home to immune cells which produce inflammatory cytokines, much like the biology of the tumor extracellular matrix.

Proteoglycans in Toll-like receptor responses and innate immunity.

The extracellular matrix (ECM) is an active and dynamic feature of tissues that not only provides gross structure but also plays key roles in cellular responses. The ever-changing microenvironment responds dynamically to cellular and external signals, and in turn influences cell fate, tissue development, and response to environmental injury or microbial invasion. It is therefore paramount to understand how the ECM components interact with each other, the environment and cells, and how they mediate their effects. Among the ECM components that have recently garnered increased attention, proteoglycans (PGs) deserve special note. Recent evidence strongly suggests that they play a crucial role both in health maintenance and disease development. In particular, proteoglycans dictate whether homeostasis or cell death will result from a given injury, by triggering and modulating activation of the innate immune system, via a conserved array of receptors that recognize exogenous (infectious) or endogenous (tissue damage) molecular patterns. Innate immune activation by proteoglycans has important implications for the understanding of cell-matrix interactions in health and disease. In this review, we will summarize the current state of knowledge of innate immune signaling by proteoglycans, discuss the implications, and explore future directions to define progress in this area of extracellular matrix biology.

Genome-wide association analysis and replication in 810,625 individuals with varicose veins

Varicose veins affect one-third of Western society, with a significant subset of patients developing venous ulceration, costing $14.9 billion annually in the USA. Current management consists of either compression stockings, or surgical ablation for more advanced disease. Most varicose veins patients report a positive family history, and heritability is ~17%. We describe the largest two-stage genome-wide association study of varicose veins in 401,656 individuals from UK Biobank, and replication in 408,969 individuals from 23andMe (total 135,514 cases and 675,111 controls). Forty-nine signals at 46 susceptibility loci were discovered. We map 237 genes to these loci, several of which are biologically plausible and tractable to therapeutic targeting. Pathway analysis identified enrichment in extracellular matrix biology, inflammation, (lymph)angiogenesis, vascular smooth muscle cell migration, and apoptosis. Using a polygenic risk score (PRS) derived in an independent cohort, we demonstrate its predictive utility and correlation with varicose veins surgery.

Identification of mechanics-responsive osteocyte signature in osteoarthritis subchondral bone.

AimsOsteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA.MethodsGene expression profiles of OA subchondral bone were used to identify disease-relevant genes and signalling pathways. RNA-sequencing data of a bone loading model were used to identify the loading-responsive gene set. Weighted gene co-expression network analysis (WGCNA) was employed to develop the osteocyte mechanics-responsive gene signature.ResultsA group of 77 persistent genes that are highly relevant to extracellular matrix (ECM) biology and bone remodelling signalling were identified in OA subchondral lesions. A loading responsive gene set, including 446 principal genes, was highly enriched in OA medial tibial plateaus compared to lateral tibial plateaus. Of this gene set, a total of 223 genes were identified as the main contributors that were strongly associated with osteocyte functions and signalling pathways, such as ECM modelling, axon guidance, Hippo, Wnt, and transforming growth factor beta (TGF-β) signalling pathways. We limited the loading-responsive genes obtained via the osteocyte transcriptome signature to identify a subgroup of genes that are highly relevant to osteocytes, as the mechanics-responsive osteocyte signature in OA. Based on WGCNA, we found that this signature was highly co-expressed and identified three clusters, including early, late, and persistently responsive genes.ConclusionIn this study, we identified the mechanics-responsive osteocyte signature in OA-lesioned subchondral bone.Cite this article: Bone Joint Res 2022;11(6):362–370.

Type XX Collagen Is Elevated in Circulation of Patients with Solid Tumors.

In the tumor microenvironment, the extracellular matrix (ECM) has been recognized as an important part of cancer development. The dominant ECM proteins are the 28 types of collagens, each with a unique function in tissue architecture. Type XX collagen, however, is poorly characterized, and little is known about its involvement in cancer. We developed an ELISA quantifying type XX collagen, named PRO-C20, using a monoclonal antibody raised against the C-terminus. PRO-C20 and PRO-C1, an ELISA targeting the N-terminal pro-peptide of type I collagen, was measured in sera of 219 patients with various solid cancer types and compared to sera levels of 33 healthy controls. PRO-C20 was subsequently measured in a separate cohort comprising 36 patients with pancreatic ductal adenocarcinoma (PDAC) and compared to 20 healthy controls and 11 patients with chronic pancreatitis. PRO-C20 was significantly elevated in all cancers tested: bladder, breast, colorectal, head and neck, kidney, lung, melanoma, ovarian, pancreatic, prostate, and stomach cancer (p < 0.01–p < 0.0001). PRO-C1 was only elevated in patients with ovarian cancer. PRO-C20 could discriminate between patients and healthy controls with AUROC values ranging from 0.76 to 0.92. Elevated levels were confirmed in a separate cohort of patients with PDAC (p < 0.0001). High PRO-C20 levels (above 2.57 nM) were predictive of poor survival after adjusting for the presence of metastasis, age, and sex (HR: 4.25, 95% CI: 1.52–11.9, p-value: 0.006). Circulating type XX collagen is elevated in sera of patients with various types of cancer and has prognostic value in PDAC. If validated, PRO-C20 may be a novel biomarker for patients with solid tumors and can help understand the ECM biology of cancer.

Proteomic Profiles Of Left Ventricular Assist Device Implantation

Introduction Left ventricular assist devices (LVADs) are widely used to support patients with advanced heart failure, but conversion from normal pulsatile flow is known to lead to vascular remodeling and ultimately may contribute to adverse vascular events. The underlying biology of such changes remains poorly understood. Hypothesis: High-throughput plasma proteomic profiling before and after LVAD implantation will identify novel biomarkers and protein mediators of LVAD cardiovascular effects. Methods Fifteen patients undergoing HeartMate II implantation at University of Colorado Hospital had serial plasma samples collected pre-LVAD (day 0) as well as days 14 and 60 after LVAD implant. Proteomic profiling was performed with 1,196 antibody-based proteomic probes on the Olink® platform. Linear mixed effects models of log2-scaled proteomic measurements, adjusted for renal function, compared protein levels between time points. GO Biological Pathway analysis was performed using both over-representation analysis (ORA) and gene set enrichment analysis (GSEA). A false discovery rate (FDR) of 5% adjusted for multiple comparisons within each analysis. Results The mean age was 57 years; 3 participants (20%) were female, 3 (20%) were Black, and 9 (60%) had a non-ischemic cardiomyopathy. At day 14, 300 proteins were significantly different from day 0 at FDR<5%. At day 60, 161 proteins differed from day 0 at FDR<5% (Figure 1a). Known biomarkers showed expected changes: B-type natriuretic peptide decreased significantly (day 14 vs day 0 β = -0.87 ± 0.26, p = 0.002; day 60 vs day 0 β = -1.19 ± 0.23, p = 1 × 10−5) as did ST2 (day 60 vs day 0 β = -0.72 ± 0.26, p = 0.01). Four proteins had statistically significant (FDR < 5%) differences at 0 vs 14 and 14 vs 60 days, highlighting novel findings (Figure 1b): CCN3, DSG3, THBS4, and COMP are all involved in extracellular matrix biology, and DSG3 and CCN3 are implicated in cell-cell communication in myocardium. Indeed, ORA and GSEA identified enrichment for blood vessel development (p = 4 × 10−6, p = 4 × 10−5, respectively) and cell adhesion (p = 9 × 10−5, p = 8 × 10−5). Conclusions Proteomic profiling identified novel protein changes following LVAD implantation, particularly among proteins involved in blood vessel development and cell adhesion. Further work is needed to understand biological implications and determine if these proteins also associate with clinical outcomes in larger cohorts and in axial compared to centrifugal flow LVADs. Left ventricular assist devices (LVADs) are widely used to support patients with advanced heart failure, but conversion from normal pulsatile flow is known to lead to vascular remodeling and ultimately may contribute to adverse vascular events. The underlying biology of such changes remains poorly understood. Hypothesis: High-throughput plasma proteomic profiling before and after LVAD implantation will identify novel biomarkers and protein mediators of LVAD cardiovascular effects. Fifteen patients undergoing HeartMate II implantation at University of Colorado Hospital had serial plasma samples collected pre-LVAD (day 0) as well as days 14 and 60 after LVAD implant. Proteomic profiling was performed with 1,196 antibody-based proteomic probes on the Olink® platform. Linear mixed effects models of log2-scaled proteomic measurements, adjusted for renal function, compared protein levels between time points. GO Biological Pathway analysis was performed using both over-representation analysis (ORA) and gene set enrichment analysis (GSEA). A false discovery rate (FDR) of 5% adjusted for multiple comparisons within each analysis. The mean age was 57 years; 3 participants (20%) were female, 3 (20%) were Black, and 9 (60%) had a non-ischemic cardiomyopathy. At day 14, 300 proteins were significantly different from day 0 at FDR<5%. At day 60, 161 proteins differed from day 0 at FDR<5% (Figure 1a). Known biomarkers showed expected changes: B-type natriuretic peptide decreased significantly (day 14 vs day 0 β = -0.87 ± 0.26, p = 0.002; day 60 vs day 0 β = -1.19 ± 0.23, p = 1 × 10−5) as did ST2 (day 60 vs day 0 β = -0.72 ± 0.26, p = 0.01). Four proteins had statistically significant (FDR < 5%) differences at 0 vs 14 and 14 vs 60 days, highlighting novel findings (Figure 1b): CCN3, DSG3, THBS4, and COMP are all involved in extracellular matrix biology, and DSG3 and CCN3 are implicated in cell-cell communication in myocardium. Indeed, ORA and GSEA identified enrichment for blood vessel development (p = 4 × 10−6, p = 4 × 10−5, respectively) and cell adhesion (p = 9 × 10−5, p = 8 × 10−5). Proteomic profiling identified novel protein changes following LVAD implantation, particularly among proteins involved in blood vessel development and cell adhesion. Further work is needed to understand biological implications and determine if these proteins also associate with clinical outcomes in larger cohorts and in axial compared to centrifugal flow LVADs.