Human Cardiac Extracellular Matrix Research Articles

Extracellular Matrix in Heart Disease: Focus on Circulating Collagen Type I and III Derived Peptides as Biomarkers of Myocardial Fibrosis and Their Potential in the Prognosis of Heart Failure: A Concise Review.

Accumulating evidence indicates that two major proteins are responsible for the structural coherence of bounding cardiomyocytes. These biomolecules are known as myocardial fibrillar collagen type I (COL1) and type III (COL3). In addition, fibronectin, laminin, fibrillin, elastin, glycoproteins, and proteoglycans take part in the formation of cardiac extracellular matrix (ECM). In physiological conditions, collagen synthesis and degradation in human cardiac ECM are well-regulated processes, but they can be impaired in certain cardiovascular diseases, such as heart failure (HF). Myocardial remodeling is part of the central mechanism of HF and involves cardiomyocyte injury and cardiac fibrosis due to increased fibrillar collagen accumulation. COL1 and COL3 are predominantly involved in this process. Specific products identified as collagen-derived peptides are released in the circulation as a result of abnormal COL1 and COL3 turnover and myocardial remodeling in HF and can be detected in patients’ sera. The role of these products in the pathogenesis of cardiac fibrosis and the possible clinical implications are the focus of numerous investigations. This paper reviews recent studies on COL1- and COL3-derived peptides in patients with HF. Their potential application as indicators of myocardial fibrosis and prognostic markers of HF is also highlighted.

Interindividual heterogeneity affects the outcome of human cardiac tissue decellularization

The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold’s intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Human Extracellular-Matrix Functionalization of 3D hiPSC-Based Cardiac Tissues Improves Cardiomyocyte Maturation.

Human induced pluripotent stem cells (hiPSC) possess significant therapeutic potential due to their high self-renewal capability and potential to differentiate into specialized cells such as cardiomyocytes. However, generated hiPSC-derived cardiomyocytes (hiPSC-CM) are still immature, with phenotypic and functional features resembling the fetal rather than their adult counterparts, which limits their application in cell-based therapies, in vitro cardiac disease modeling, and drug cardiotoxicity screening. Recent discoveries have demonstrated the potential of the extracellular matrix (ECM) as a critical regulator in development, homeostasis, and injury of the cardiac microenvironment. Within this context, this work aimed to assess the impact of human cardiac ECM in the phenotype and maturation features of hiPSC-CM. Human ECM was isolated from myocardium tissue through a physical decellularization approach. The cardiac tissue decellularization process reduced DNA content significantly while maintaining ECM composition in terms of sulfated glycosaminoglycans (s-GAG) and collagen content. These ECM particles were successfully incorporated in three-dimensional (3D) hiPSC-CM aggregates (CM+ECM) with no impact on viability and metabolic activity throughout 20 days in 3D culture conditions. Also, CM+ECM aggregates displayed organized and longer sarcomeres, with improved calcium handling when compared to hiPSC-CM aggregates. This study shows that human cardiac ECM functionalization of hiPSC-based cardiac tissues improves cardiomyocyte maturation. The knowledge generated herein provides essential insights to streamline the application of ECM in the development of hiPSC-based therapies targeting cardiac diseases.

Towards a Novel Patch Material for Cardiac Applications: Tissue-Specific Extracellular Matrix Introduces Essential Key Features to Decellularized Amniotic Membrane.

There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining it with cell-free amniotic membrane via a dry-coating procedure. Cardiac biocompatibility and immunogenicity were tested in vitro using human cardiac fibroblasts, epicardial progenitor cells, murine HL-1 cells, and human immune cells derived from buffy coat. Processing of the ECM preserved important matrix proteins as demonstrated by mass spectrometry. ECM coating did not alter the mechanical characteristics of decellularized amniotic membrane but did cause a clear increase in adhesion capacity, cell proliferation and viability. Activated monocytes secreted less pro-inflammatory cytokines, and both macrophage polarization towards the pro-inflammatory M1 type and T cell proliferation were prevented. We conclude that the incorporation of human cardiac ECM hydrogel shifts and enhances the bioactivity of decellularized amniotic membrane, facilitating its use in future cardiac applications.

Cartilage intermediate layer protein 1 (CILP1): A novel mediator of cardiac extracellular matrix remodelling

Heart failure is accompanied by extracellular matrix (ECM) remodelling, often leading to cardiac fibrosis. In the present study we explored the significance of cartilage intermediate layer protein 1 (CILP1) as a novel mediator of cardiac ECM remodelling. Whole genome transcriptional analysis of human cardiac tissue samples revealed a strong association of CILP1 with many structural (e.g. COL1A2 r2 = 0.83) and non-structural (e.g. TGFB3 r2 = 0.75) ECM proteins. Gene enrichment analysis further underscored the involvement of CILP1 in human cardiac ECM remodelling and TGFβ signalling. Myocardial CILP1 protein levels were significantly elevated in human infarct tissue and in aortic valve stenosis patients. CILP1 mRNA levels markedly increased in mouse heart after myocardial infarction, transverse aortic constriction, and angiotensin II treatment. Cardiac fibroblasts were found to be the primary source of cardiac CILP1 expression. Recombinant CILP1 inhibited TGFβ-induced αSMA gene and protein expression in cardiac fibroblasts. In addition, CILP1 overexpression in HEK293 cells strongly (5-fold p < 0.05) inhibited TGFβ signalling activity. In conclusion, our study identifies CILP1 as a new cardiac matricellular protein interfering with pro-fibrotic TGFβ signalling, and as a novel sensitive marker for cardiac fibrosis.

Glycoproteomics Reveals Decorin Peptides With Anti-Myostatin Activity in Human Atrial Fibrillation.

Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

The cytoprotective capacity of processed human cardiac extracellular matrix.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66μm (15-157μm) by laser diffraction, and 57μm (20-182μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39±12%, P<0.05) and proliferation (16±3%, P<0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240±32%; BrdU uptake, 45±14%; LDH release, -72±7%; P<0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25°C, 1mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Decellularized human cardiac extracellular matrix as a natural scaffold for stem cell-based cardiac engineering

Decellularized human cardiac extracellular matrix as a natural scaffold for stem cell-based cardiac engineering

Novel xeno-free human heart matrix-derived three-dimensional scaffolds.

RationaleMyocardial infarction (MI) results in damaged heart tissue which can progress to severely reduce cardiac function, leading to death. Recent studies have injected dissociated, suspended cardiac cells into coronary arteries to restore function with limited results attributed to poor cell retention and cell death. Extracellular matrix (ECM) injected into damaged cardiac tissue sites show some promising effects. However, combined use of human cardiac ECM and cardiac cells may produce superior benefits to restore cardiac function.ObjectiveThis study was designed to assess use of new three-dimensional human heart ECM-derived scaffolds to serve as vehicles to deliver cardiac-derived cells directly to damaged heart tissue and improve cell retention at these sites while also providing biomechanical support and attracting host cell recruitment.Methods and ResultsECM-derived porous protein scaffolds were fabricated from human heart tissues. These scaffolds were designed to carry, actively promote and preserve cardiac cell phenotype, viability and functional retention in tissue sites. ECM scaffolds were optimized and were seeded with human cardiomyocytes, cultured and subsequently implanted ex vivo onto infarcted murine epicardium. Seeded human cardiomyocytes readily adhered to human cardiac-derived ECM scaffolds and maintained representative phenotypes including expression of cardiomyocyte-specific markers, and remained electrically synchronous within the scaffold in vitro. Ex vivo, cardiomyocyte-seeded ECM scaffolds spontaneously adhered and incorporated into murine ventricle.ConclusionsDecellularized human cardiac tissue-derived 3D ECM scaffolds are effective delivery vehicles for human cardiac cells to directly target ischemic heart tissue and warrant further studies to assess their therapeutic potential in restoring essential cardiac functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0559-0) contains supplementary material, which is available to authorized users.

(389) - Functional and Biochemical Assessment of Porcine Hearts After Simulated Donation After Circulatory Death

Purpose: Heart failure results from damaged myocardium that can severely reduce function and lead to death with very few options for treatment. Recent studies have injected stem cells into the coronary arteries to improve function. Though the potential of stem cell therapy is great, only limited efficacy has been attained, Extracellular matrix (ECM) proteins derived ECM from bowel or bladder and stem cells in concert may have superior benefits to either alone but the homing cues are still limited for engraftment due to lack of organ specific signaling and xenogenic issues. Our goal was to derive a novel three-dimensional human heart extracellular matrix (ECM)-derived scaffold that could serve as a vehicle to deliver cardiac or stem cells directly to the damaged tissue of the heart and remain at the treatment site. Methods: Scaffolds were created from purified human heart ECM proteins to expedite clinical translation and actively promote and preserve heart cell phenotype. Scaffolds were imparted with pores 250 um in size to enhance diffusion and cell penetration and distribution. Human cardiomyocytes and cardiac derived-iPSCs were seeded into scaffolds 3 mm in diameter and 0.3 mm thick, cultured and subsequently implanted into NOD-SCID mice. Results: The human cardiomyocytes and cardiac derived iPSCs readily adhered to human cardiac-derived ECM protein scaffolds. Cardiomoycytes within the scaffold maintained representative phenotypes including expression of cardiomyocyte-specific markers and remained electrically active, even beating the scaffold in unison in vitro. In vivo, cardiomyocyte-seeded scaffolds spontaneously adhered to and incorporated with murine infarcted tissue. Conclusion: These results indicate that a novel 3D scaffold made of purified human cardiac ECM can be used as a delivery vehicle for human cardiac cells to directly target damaged heart tissue. Optimization of scaffolds and cell interactions need to be studied further in vivo.

(390) - Three-Dimensional Human-Heart Derived Scaffolds

Heart failure results from damaged myocardium that can severely reduce function and lead to death with very few options for treatment. Recent studies have injected stem cells into the coronary arteries to improve function. Though the potential of stem cell therapy is great, only limited efficacy has been attained, Extracellular matrix (ECM) proteins derived ECM from bowel or bladder and stem cells in concert may have superior benefits to either alone but the homing cues are still limited for engraftment due to lack of organ specific signaling and xenogenic issues. Our goal was to derive a novel three-dimensional human heart extracellular matrix (ECM)-derived scaffold that could serve as a vehicle to deliver cardiac or stem cells directly to the damaged tissue of the heart and remain at the treatment site. Scaffolds were created from purified human heart ECM proteins to expedite clinical translation and actively promote and preserve heart cell phenotype. Scaffolds were imparted with pores 250 um in size to enhance diffusion and cell penetration and distribution. Human cardiomyocytes and cardiac derived-iPSCs were seeded into scaffolds 3 mm in diameter and 0.3 mm thick, cultured and subsequently implanted into NOD-SCID mice. The human cardiomyocytes and cardiac derived iPSCs readily adhered to human cardiac-derived ECM protein scaffolds. Cardiomoycytes within the scaffold maintained representative phenotypes including expression of cardiomyocyte-specific markers and remained electrically active, even beating the scaffold in unison in vitro. In vivo, cardiomyocyte-seeded scaffolds spontaneously adhered to and incorporated with murine infarcted tissue. These results indicate that a novel 3D scaffold made of purified human cardiac ECM can be used as a delivery vehicle for human cardiac cells to directly target damaged heart tissue. Optimization of scaffolds and cell interactions need to be studied further in vivo.

Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells†

Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell attachment, apoptosis induction (Caspase 3/7 activity) and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium conversion) were followed. Transcriptional activation of genes involved in pluripotency; early and late myocardial development; and endothelial, ectodermal or endodermal commitment were monitored by quantitative real-time polymerase chain reaction (rtPCR). Protein expression of selected markers was confirmed by immunohistology. cECM supported the proliferation of ESCs and iPSCs, and Caspase 3/7 activity was significantly lower compared with standard culture. Cardiac lineage commitment was favoured when ESCs or iPSCs were grown on cECM, as evidenced by the significantly increased mRNA expression of cardiac alpha myosin heavy polypeptide 6 (Myh6), cardiac troponin T2 (Tnnt2) and NK2 homeobox 5 (Nkx2.5) as well as positive immunohistology for cardiac troponin T and heavy-chain cardiac myosin protein. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSCs showed no evidence of cardiomyocyte differentiation. Human cardiac ECM seems to direct differentiation of pluripotent stem cells towards a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair, and may ultimately increase the therapeutic efficacy of cell therapy in heart failure patients.

Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium

Extracellular matrix (ECM) derived by tissue decellularization has applications as a tissue engineering scaffold and for support of cellular regeneration. Myocardial ECM from animals has been produced by whole-organ perfusion or immersion processes, but methods for preparation of human myocardial ECM for therapy and research have not been compared in detail, yet. We analyzed the impact of decellularization processes on human myocardial ECM, and tested its ability to serve as a scaffold for cell seeding. Sodium dodecyl sulfate (SDS)-based decellularization, but not treatments based on Triton X-100, deoxycholate or hypo/hypertonic incubations, removed cells satisfactorily, and incubation with fetal bovine serum (FBS) eliminated residual DNA. ECM architecture was best preserved by a protocol consisting of 2 h lysis, 6 h SDS, and 3 h FBS, but age and pathology of the donor tissue are highly important for producing reproducible, high-quality scaffolds. We also studied ECM repopulation with mesenchymal stem cells (CB-MSC), cardiomyocytes derived from induced pluripotent stem cells (iPS-CM), and na€ıve neonatal mouse cardiomyocytes. Cells attached to the matrix and proliferated and displayed higher viability than in standard culture. We conclude that human cardiac ECM sheets may be suitable scaffold for cell-matrix interaction studies and as a biomaterial for tissue regeneration and engineering.

Crosstalk between human cardiac extracellular matrix and cardiomyocyte-like cells

Objectives: Extracellular matrix (ECM) has been shown to influence differentiation, survival, and integration of stem and progenitor cells and might also play a key role in the outcome of cardiac cell therapy. In order to directly study the crosstalk between ECM and progenitor cells in the human heart, we recellularized ECM that was obtained via decellularization with different cell types and studied their integration capacity.

Interactions between myocardial extracellular matrix and somatic and pluripotent stem cells- Implications for cardiac cell therapy

Objective: Cardiac regenerative medicine still relies on the delivery of exogenous cells to the diseased heart. When injected into the myocardium, cells interact with the surrounding extracellular matrix (ECM), which may influence cell proliferation, survival, integration and differentiation. We sought to directly observe cell-to-matrix interactions by generating cell-free human cardiac ECM and used this matrix as a scaffold for seeding with stem- and progenitor cells.