Human myxomatous mitral valve prolapse: role of bone morphogenetic protein 4 in valvular interstitial cell activation

Abstract

Myxomatous mitral valve prolapse (MVP) is the most common cardiac valvular abnormality in industrialized countries and a leading cause of mitral valve surgery for isolated mitral regurgitation. The key role of valvular interstitial cells (VICs) during mitral valve development and homeostasis has been recently suggested, however little is known about the molecular pathways leading to MVP. We aim to characterize bone morphogenetic protein 4 (BMP4) as a cellular regulator of mitral VIC activation towards a pathologic synthetic phenotype and to analyze the cellular phenotypic changes and extracellular matrix (ECM) reorganization associated with the development of myxomatous MVP. Microarray analysis showed significant up regulation of BMP4-mediated signaling molecules in myxomatous MVP when compared to controls. Histological analysis and cellular characterization suggest that during myxomatous MVP development, healthy quiescent mitral VICs undergo a phenotypic activation via up regulation of BMP4-mediated pathway. In vitro hBMP4 treatment of isolated human mitral VICs mimics the cellular activation and ECM remodeling as seen in MVP tissues. The present study characterizes the cell biology of mitral VICs in physiological and pathological conditions and provides insights into the molecular and cellular mechanisms mediated by BMP4 during MVP. The ability to test and control the plasticity of VICs using different molecules may help in developing new diagnostic and therapeutic strategies for myxomatous MVP.

Figure 1. Macroscopic and Histological Analysis of myxomatous mitral valve and control

[A] Comparison between normal mitral valve and patient with P2 prolapse and mitral regurgitation: (a) 3D Echocardiographic appearance of normal mitral valve with no evidence of prolapse, (b) Normal mitral valve in cadaver, (c) 3D echocardiographic appearance of P2 prolapse (marked by asterisk *) in patient with mitral regurgitation (d) intra-operative videoscopic view of prolapsed P2 scallop prior to resection. [B] Composite photomicrographs of sections of human mitral valve leaflets (transverse sections). Controls (a and b), and diseased valves (c and d) high-definition images have been assembled to rebuild the leaflet structures. H&E (a, c) and Movat Pentachrome staining (b, d) are reported. FE: free edge; VS: ventricularis, AtS: Atrialis [C] (a) Representative image of H&E section of the mitral valve spongiosa layers of control and MVP patient and relative number of VICs per field, (b) quantification of total cell number within selected fields of the spongiosa layers. Bar graph: 50 μm.

Figure 2. Microarray Analysis

[A] Relative expression of mRNA transcripts over the total number of genes included in the microarray analysis. ECM organization, cell-cell and cell-matrix adhesion, growth factors and inflammatory response are reported. [B] BMP4 related VIC activation- and BMP4-mediated Osteogenesis induced gene transcripts up regulated or down regulated in the microarray analysis.

Figure 3. Graph bar representation of mRNA variations in myxomatous MVP vs. controls

qPCR analysis were performed for 15 MVP and 15 control tissues. Relative transcript abundance of 18S-normalized PCR products is given, in which the control abundance of gene is assigned a value of 1. Data expressed as RQ ±dCT SE. [A] BMP4-upregulated VIC activation markers, [B] ECM remodeling markers, and [C] BMP4-mediated osteogenesis makers were analyzed as described (* denotes p<0.05).

Figure 4. BMP4-mediated pathways and ECM reorganization in Myxomatous MVP and controls

[A] Immunohistochemistry of surgically resected myxomatous P2 prolapse and controls as indicated. 4 μm thick Cross sections; 63X Magnification, scale bar: 16μm. [B]. Shows western blot (a) and densitometry analysis (b) for expression of BMP4, Sox9 and ERK ½ (loading controls) in tissue lysates from controls and MVP leaflets (* denotes p<0.05). [C] H&E, Masson’s Trichrome (MT), Movat Pentachrome and Biglycan staining to visualize the extracellular structure, Valvular interstitial cell content, proteoglycans, collagen and elastin composition of the three valvular layers. Magnification 10X, Scale 100 μm.

Figure 5. Characterization of Mitral VICs

[A] Immunofluorescence staining for Vimentin, SMAα2, and Desmin in the isolated VICs. Magnification: 40X, Scale: 50μm. [B] Relative abundance of transcripts for Vimentin, SMAα2 and SM22α in MVP-derived VICs, using qPCR. (* denotes p<0.05). [C] Western blots showing expression of SMAα2, desmin and ERK1/2 (loading control) [D] Schematic representation for expression of VIC markers.

Figure 6. In vitro BMP4 treatment

BMP4 regulated transcripts were detected after 12 or 21 days of BMP4 treatments of MV-derived cells from patients and controls. (A) BMP4 signaling molecules upregulated by BMP4 treatment in the MVP-derived VICs. (B) BMP4 mediated changes in ECM remodeling genes: Up regulation of Col3A1, Biglycan and DCN in MVP-isolated VICs was seen. An early up regulation of Col2A1 and Biglycan in control-isolated cells was observed. Relative transcript abundance of 18S-normalized PCR products is given, in which the control abundance of gene is assigned a value of 1. Data expressed as RQ±dCT SE. (C) BMP4-related osteogenesis markers No significant differences were noted on both cell populations. (D) Morphological analysis of MV-derived VIC from patients and controls. BMP4 treatment induces morphological changes on MVP-derived cells, but not in control-derived cells. Magnification: 10X, Scale bar 100μm (* denotes p<0.05).

Figure 7. Human Myxomatous Mitral Valve Prolapse Tissue: Role of Bone Morphogenetic Protein 4 in valvular interstitial cell activation and Extracellular Matrix Homeostasis

(A) Schematic representation of the role of BMP4 in cellular activation and ECM remodeling in Myxomatous MVP leaflet and control. Ats: Atrialis; V: ventricularis (B) Table of cellular and extracellular changes associated with the increased levels of BMP4 in Myxomatous MVP and Controls.