Pathological ECM remodelling and biomineralization in human aortic valve and bioprosthesis tissue were investigated by Fourier transformed infrared (FT-IR) spectroscopic imaging and multivariate data analysis. Results of histological von Kossa staining to monitor hydroxyapatite biomineralization correlated to the definition of mineralized tissue using FT-IR spectroscopic imaging. Spectra exhibit signals of carbonate and phosphate groups of hydroxyapatite. Proteins could be identified by the amide I and amide II bands. Proteins were detected in the calcified human aortic valve tissue, but no absorption signals of proteins were observed in the mineralized bioprosthesis sample region. A shift of the amide I band from 1654 cm−1 to 1636 cm−1 was assumed to result from β-sheet structures. This band shift was observed in regions where the mineralization process had been identified but also in non-mineralized bioprosthesis tissue independent of prior implantation. The increased occurrence of β-sheet conformation is hypothesized to be a promoter of the biomineralization process. FT-IR spectroscopic imaging offers a wealth of chemical information. For example, slight variations in band position and intensity allow investigation of heterogeneity across aortic valve tissue sections. The exact evaluation of these properties and correlation with conventional histological staining techniques give insights into aortic valve tissue remodelling and calcific pathogenesis.
Introduction The involvement of the bone marrow microenvironment (BMME) into disease progression and therapeutic response of myelodysplastic syndromes (MDS) is indisputable. Hereby, mesenchymal stromal cells (MSCs) play an important role for both the support of the leukemic clone and the remaining healthy hematopoietic stem and progenitor cells (HSPCs). The extracellular matrix (ECM) secreted by MSCs regulates stem cell fate through the modulation of cytokine and growth factor delivery and may also be targeted by clinically available drugs such as luspatercept, a novel recombinant fusion protein containing modified extracellular domain of activin receptor IIB. Luspatercept is a first-in-class erythroid maturation agent with promising results in lower-risk MDS patients with red blood cell transfusion dependency. Aim To shed light on the largely unknown composition and function of the MSC-derived ECM, we have characterized ECM from MDS patients vs. healthy controls and elucidate how luspatercept may modulate their functional characteristics. Methods Bone marrow-derived MSCs from patients with lower-risk MDS and age-matched healthy donors (HD) were treated with RAP-536, a murine homologue of luspatercept harboring the same activin receptor IIB domain. MSCs of three patients were treated with RAP-536 and RNA sequencing was carried out. Gene expression and pathway analyses were performed using the Reactome tool (https://reactome.org). Candidate genes were validated by quantitative real-time PCR (qPCR). For the generation of ECM, MSCs were seeded on poly-octadecene-alt-maleic anhydride and human fibronectin coated glass slides in the presence or absence of RAP-536. To yield cell-free ECM structures, cultures were decellularized at day 10 and analyzed by scanning electron microscopy (SEM), sulfated glycosaminoglycan (GAG), fibronectin and collagen staining as well as GAG quantification (Blyscan assay). Moreover, purified HD CD34+ HSPCs were cultured on ECM scaffolds for 6 and 9 days, respectively. Subsequently, expansion of adherent and supernatant cells was determined and the phenotype was analyzed by flow cytometry. Results RNA sequencing of MDS MSCs after six days of RAP-536 treatment revealed a total of 58 significantly regulated genes, thereof 24 up- and 34 down-regulated genes. Gene enrichment and pathway analyses revealed a striking involvement in ECM organization, collagen biosynthesis and formation. Moreover, integrin cell surface interaction genes showed significantly differential expression. Focusing on collagens as important ECM components, we identified Col7A1 and Col4A2 to be down-regulated. Indeed, both collagen mRNAs were significantly decreased by 46% and 25%, respectively, in MSCs after RAP-536 treatment compared to untreated controls. SEM characterization and immunofluorescence staining of the ECM showed a more compact fiber network produced by MDS MSCs. Moreover, MDS ECM contained higher levels of collagen and GAGs. Blyscan assay confirmed the latter observation, showing significantly higher sulfated GAG concentrations in MDS ECM. Interestingly, trapping of TGFβ superfamily ligands, such as GDF-11, by RAP-536 clearly reduced Col4 staining intensity in MDS MSC ECM. Structural and compositional ECM differences had functional impact on the expansion of HSPCs cultured on the matrices. Significant higher total cell numbers were detected on healthy ECM (18.3-fold vs. 12.1-fold expansion, *p< 0.05) but not on MDS ECM (12.9-fold) after 9 days of culture. The number of adherent cells increased 8.5-fold on healthy and 4.3-fold on MDS ECM and could be further increased after RAP-536 treatment of MSCs. Using flow cytometry, we found a 3.1-fold increased proportion of CD90+ HSPCs in the adherent fraction on healthy but only 1.8-fold on MDS MSC ECM. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be significantly higher expressed in the adherent HSPC fraction. RAP-536 treatment resulted in up to 20% higher expression of both CD90 and integrin subunits. Summary We demonstrate an association between induced collagen abundance and reduced hematopoietic support in ECM derived from MDS MSCs and conclude that compact MDS ECM structure induced by TGFβ superfamily members may alter the cytokine environment for HSPCs. Consequently, TGFβ ligand trapping by RAP-536/luspatercept leads to ECM re-organization and thus an improved hematopoietic support. Disclosures Stoelzel: Shire: Consultancy, Other: Travel funding; Neovii: Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.
The degeneration of heart valve bioprostheses due to calcification processes is caused by the intercalation of calciumhydroxyapatite in pericardium collagen bundles. Variations of the protein secondary structure of biomaterials according to preparation are relevant for this mineralization process and thus the structural characterization of innovative bioprostheses materials is of great importance. The gold standard for prostheses preparation is glutaraldehyde (GA)-fixation of bovine pericardium that adversely promotes calcification. The novel GA-free SULEEI-treatment of bovine pericardium includes decellularization, UV-crosslinking, and electron beam sterilization. The aim of this study is the structural characterization of SULEEI-treated and GA-fixed bovine pericardium. IR spectroscopic imaging combined with multivariate data and curve fit analysis was applied to investigate the amide I and amide II regions of SULEEI-treated and GA-fixed samples. The spectroscopic images of GA-fixed pericardial tissue exhibited a generally high content of amine groups and side chains providing nucleation points for calcification processes. In contrast, in SULEEI-treated tissue, the typical α-helical structure was retained and was supposed to be less prone to deterioration.
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