Nonbiased Molecular Screening Identifies Novel Molecular Regulators of Fibrogenic and Proliferative Signaling in Myxomatous Mitral Valve DiseaseCLINICAL PERSPECTIVE
Background—Pathological processes underlying myxomatous mitral valve degeneration (MMVD) remain poorly understood. We sought to identify novel mechanisms contributing to the development of this condition.
Methods and Results—Microarrays were used to measure gene expression in 11 myxomatous and 11 nonmyxomatous human mitral valves. Differential gene expression (thresholds P<0.05; fold-change >1.5) and pathway activation (Ingenuity) were confirmed using quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Contributions of bone morphogenetic protein 4 and transforming growth factor (TGF)-β2 to differential gene expression were evaluated in vitro. Contributions of angiotensin II to differential pathway activation were examined in mice in vivo. A total of 2602 genes were differentially expressed between myxomatous and nonmyxomatous valves. Canonical TGF-β signaling was increased in MMVD because of increased ligand expression and derepression of SMA mothers against decapentaplegic 2/3 signaling and was confirmed with quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Myxomatous valves demonstrated activation of canonical bone morphogenetic protein and Wnt/β-catenin signaling and upregulation of their common target runt-related transcription factor 2. Our data set provided transcriptional and immunohistochemical evidence for activated immune cell infiltration. In vitro treatment of mitral valve interstitial cells with TGF-β2 increased β-catenin signaling at mRNA and protein levels, suggesting interactions between TGF-β2 and Wnt signaling. In vivo infusion of mice with angiotensin II recaptured several changes in signaling pathways characteristic of human MMVD.
Conclusions—These data support a new disease framework whereby activation of TGF-β2, bone morphogenetic protein 4, Wnt/β-catenin, or immune signaling plays major roles in the pathogenesis of MMVD. We propose these pathways act in a context-dependent manner to drive phenotypic changes that fundamentally differ from those observed in aortic valve disease and open novel avenues guiding future research into the pathogenesis of MMVD.
Myxomatous mitral valve degeneration (MMVD) has an estimated prevalence of 2% to 3% in the general population.1 Abnormal thickening of valve leaflets alongside thickening, thinning, or elongation of the chordae tendinae are hallmark features of MMVD.2,3 The resultant disruption of the structural integrity of the valvular apparatus leads to the eventual development of mitral valve prolapse (MVP). In the absence of appropriate and timely intervention, MVP secondary to MMVD sequentially progresses to mitral regurgitation, impaired left ventricular (LV) function and heart failure, and ultimately death.1,4
Clinical Perspective on p 528
Surgical strategies remain the therapeutic gold standard in MMVD,5 and normal life expectancy can be restored after mitral valve repair.6–8 Molecular mechanisms contributing to MMVD remain poorly understood, however, and have been largely confined to the role of transforming growth factor-β1 (TGF-β1). Upregulation of TGF-β1 has been reported in animals9,10 and humans with MVP11,12 and is thought to contribute to extracellular matrix remodeling and fibrosis.12,13 Interestingly, hypertension is a major risk factor for accelerated progression of MMVD,14 and a recent report implicated angiotensin II (AngII) in the activation of TGF-β signaling in valvular interstitial cells in vitro.11 To date, however, identification of alternative and parallel pathways outside of TGF-β1 signaling that contribute to initiation or progression of MMVD has been limited.
To address this issue, we used comprehensive whole genome expression arrays as a nonbiased method to identify novel signaling pathways altered in myxomatous versus nonmyxomatous human mitral valves. To validate the relative importance and contribution of some of the identified pathways in the development of MMVD, we performed directed in vitro experiments in human/mouse mitral valve interstitial cells (MVICs), as well as the molecular and phenotypic consequences of in vivo AngII infusion in murine mitral valve tissue. Our over-riding goals were to identify novel mechanisms contributing to the development of MMVD and to determine the extent to which an experimental mouse model can recapture molecular and phenotypic aberrations characteristics of MMVD.
Nonbiased Identification of Novel Signaling Pathways in MMVD
Affymetrix Human Genome U133 Plus 2.0 expression arrays were used to measure differential gene expression in a random sample of 11 myxomatous and 11 nonmyxomatous human mitral valves obtained during surgeries at Mayo Clinic in Rochester, MN (see Data Supplement). Mayo Clinic’s Institutional Review Board approved the current study, and valid informed consent was obtained for all patients.
Confirmation of Differentially Regulated Signaling Pathways in MMVD
Differential expression of key pathway genes between myxomatous and nonmyxomatous human mitral valves was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR; see Table I in the Data Supplement for gene expression primers used) and immunohistochemistry (see Data Supplement for details).
In Vitro Model of MMVD
Human and murine (C57BL/6J) MVICs were harvested and cultured, and subsequently treated with exogenous bone morphogenetic protein 4 (BMP4), TGF-β2, or control saline for 24 hours. Consequent molecular changes were identified using qRT-PCR, Western blotting, and immunocytochemistry (see Data Supplement).
In Vivo Model of MMVD
Young C57BL/6J mice (aged 2–3 months) were treated with pressor doses of AngII or saline via osmotic minipump for 14 days. Molecular changes in mitral valves were assessed by qRT-PCR and immunohistochemistry. Echocardiography was used to assess mitral valve and LV function on day 14 postminipump implantation (see Data Supplement). All protocols pertaining to animal studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee and conformed to guidelines set forth by the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals.
Differential gene expression between myxomatous and nonmyxomatous mitral valves was determined by linear modeling using t test, with significance thresholds of a 1.5-fold change in expression and a P value <0.05. Secondary analysis adjusting for multiple comparisons was performed using the false discovery rate statistical approach. Adjusted P values (q values) were obtained and 2 commonly used significance thresholds of q<0.05 and q<0.1 were applied. Ingenuity pathway analysis systems were used to identify differentially regulated signaling pathways (Ingenuity Systems, Inc).
Results of confirmatory qRT-PCR and immunohistochemical staining were compared between myxomatous and nonmyxomatous human mitral valves using Student t test. Linear regression and coefficients of determination (R2) were used to depict correlations between microarray and qRT-PCR–determined gene expression for all validated genes (Table II in the Data Supplement).
Differential gene expression (qRT-PCR) and protein levels (immunohistochemistry) between in vivo saline versus AngII-treated mouse mitral valves were compared using Student t tests. Results of qRT-PCR and Western blotting experiments comparing effects of in vitro treatment with TGF-β2 or BMP4 to control conditions (saline/vehicle) were analyzed using Dunnett test for multiple comparisons. Significance thresholds of P<0.05 were used throughout. For further details, see expanded Statistical Methods in the Data Supplement.
Differential Gene Expression in MMVD
Patient-specific data including demographics, surgical indication and baseline medications are outlined in Tables III to V in the Data Supplement. From our primary microarray analyses, 2602 unique genes were differentially expressed between myxomatous and nonmyxomatous human mitral valves (1454 upregulated, 1148 downregulated; Figure I in the Data Supplement and Table VI in the Data Supplement). From our secondary microarray analyses, 2125 unique genes were differentially expressed between myxomatous and nonmyxomatous human mitral valves (1317 upregulated, 808 downregulated) with a significance threshold of q <0.05. When using a significance threshold of q <0.1, all 2602 genes from our primary analysis retained statistical significance (q values in Table VI in the Data Supplement). Because ingenuity pathway analyses using significance thresholds of q <0.05 and <0.1 yielded near identical results for key differentially regulated signaling cascades, the subsequent data present herein are derived from the primary analysis data set.
TGF-β Signaling and Gene Expression Are Increased in MMVD
Microarray pathway analyses identified significantly dysregulated expression of genes associated with canonical TGF-β signaling in myxomatous mitral valves (Figure 1A). More precisely, TGF-β2 ligand, intranuclear transcription factors (Runt-related transcription factor 2 [Runx2]), coactivators (cyclic-AMP-responsive element-binding protein 5), and profibrotic gene products (collagen type I, αI, matrix metalloproteinases 2 and 16, fibroblast activation protein, fibronectin, and Fascin) were also increased in myxomatous valves. Expression of the decoy receptor BMP and activin membrane-bound inhibitor homolog (Xenopus laevis) (BAMBI), intracellular inhibitors of TGF-β signaling (salt-inducible kinase 1 [SIK1]), and transcriptional repressors (c-Myc, TGF-β-induced factor) were reduced in myxomatous tissue. qRT-PCR confirmed significant changes in TGF-β2 ligand (Figure 1B), BAMBI (Figure 1C), and SIK1 (Figure 1D; all P<0.05). Figure 1E depicts a working model of these changes in the context of canonical TGF-β signaling.
Multiple Classically Osteogenic Signaling Pathways Are Increased in MMVD
Increased BMP signaling in myxomatous mitral tissue was evident from the microarray screen (Figure 2A), as expression of BMP4, transcriptional coactivators (cyclic-AMP-responsive element-binding protein 5) and BMP target genes (Runx2, Wnt-inducible pathway protein 1 [WISP1]) was increased in myxomatous valves. In contrast, intracellular signaling inhibitors (transducer of avian erythroblastosis oncogene B-2 [erbB2]) and transcriptional corepressors (c-Myc, TGF-β–induced factor) were reduced in diseased mitral valves. qRT-PCR confirmed changes in BMP4 ligand (Figure 2B) and Runx2 (Figure 2C) in myxomatous versus nonmyxomatous tissue (both P<0.05). Immunohistochemical staining demonstrated increases in the canonical intracellular signaling protein pSMA mothers against decapentaplegic (SMAD) 1/5/8 (Figure 2D and 2E; Figure IIA in the Data Supplement). Figure 2F outlines a working model for contributions of these changes toward BMP signaling.
Microarray pathway analyses also suggested that canonical Wnt/β-catenin signaling was increased in valve tissue from patients with MMVD (Figure 3A). Levels of Wnt ligand (Wnt9A) and receptor (frizzled 8), extracellular positive modulators of Wnt signaling (R-Spondin 2, Norrie disease [pseudoglioma]), intranuclear transcription factors (transcription factor 4), and classically procalcific gene products (Runx2, WISP1) were increased in MMVD. In contrast, transcription repressors (HMG-box transcription factor 1) and corepressors (transducer-like enhancer of split 1) were reduced in MMVD. qRT-PCR confirmed increases in Wnt9A, frizzled 8, R-Spondin 2, and WISP1 (Figure 3B–3E; all P<0.05). Figure 3F delineates a model representation of these changes in relation to Wnt signaling.
Evidence of Immune Cell Infiltration and Activation in Myxomatous Mitral Valves
There was evidence of immune cell infiltration and activation in myxomatous versus nonmyxomatous mitral valve tissue (Figure 4A). Cell-surface receptors cluster of differentiation (CD) 14 and CD83 alongside cytokines (interleukin-7, chemokine (C-X3-C motif) ligand 1) and cytokine receptors (chemokine (C-X3-C motif) receptor 1) were increased in MMVD. Levels of toll-like receptors 3 and 7 were also increased in myxomatous tissue. Upregulation of CD14, CD83, and chemokine (C-X3-C motif) receptor 1 in myxomatous valves was corroborated by qRT-PCR (Figure 4B–4D; all P<0.05). Immunohistochemical staining for anti-CD14 antibodies additionally verified abnormally increased infiltration of immune cells in myxomatous valves (Figure 4E; Figure XIIB in the Data Supplement). Immune localization in mitral tissue seemed independent of interleukin-6, a cytokine commonly used as a marker of inflammation, which had an ≈17-fold reduction in expression in myxomatous valves (Figure 4A, see Full Microarray Data Set in the Data Supplement). Figure 4F depicts a working model for these transcriptional changes as relating to immune activation and infiltration.
Evidence of Increased Cellular Proliferation in MMVD
From the microarray data set, we identified reduced expression of the cell-cycle inhibitor CDKN1A (see Full Microarray Data Set in the Data Supplement) in myxomatous valves, confirmed by qRT-PCR (Figure IIIA in the Data Supplement). Reciprocally, we used immunohistochemistry to demonstrate upregulation of the proliferation marker proliferation cell nuclear antigen (PCNA) in myxomatous valves (Figures IIC, IID, IIIB, and IIIC in the Data Supplement).
Molecular Cross-Talk in MMVD
Figure 5 depicts the network of potential protein–protein interactions between molecules differentially expressed in myxomatous versus nonmyxomatous human mitral valves, and their neighboring interactors, derived using PathwayLinker.15 The specific goal of this figure is to highlight numerous interactions between key molecules that were identified as differentially regulated in our microarray analysis (ie, TGF-β signaling, BMP signaling, Wnt/β-catenin signaling, immune-related processes) and thereby provide an initial framework to guide future investigation into complex molecular interactions contributing to the pathogenesis of MMVD.
Effects of In Vitro Treatment With Exogenous TGF-β2 and BMP4 on Profibrotic, Procalcific, and Immune-Related Signaling Responses in Nonmyxomatous MVICs
TGF-β2 treatment increased protein levels of pSMAD2 on Western blot analysis (Figure 6A; Figure IVA in the Data Supplement) although immunocytochemical changes failed to show significant differences (Figure 6B; Figure VA in the Data Supplement). TGF-β2 increased mRNA levels of the fibrogenic target gene collagen type I, αI (Figure 6C), and expression of intracellular TGF-β signaling inhibitors SIK1 (Figure 6D), SMAD-specific E3 ubiquitin ligase 1 and SMAD-specific E3 ubiquitin ligase 2 (Figure VIA and VIB in the Data Supplement) in human MVICs. Exogenous treatment of human MVICs with TGF-β2 yielded significant reductions in expression of the decoy receptor BAMBI (Figure VIC in the Data Supplement). Conversely, after BMP4 treatment, BAMBI expression was increased (Figure VIC in the Data Supplement) in the absence of other visible alterations in TGF-β signaling (Figure 6A–6D). With the exception of unchanged BAMBI expression after BMP4 treatment, similar changes were observed in murine MVICs (Figures VII–IX and XA in the Data Supplement).
Considering BMP-network genes, in vitro TGF-β2 treatment did not significantly alter pSMAD1/5/8 protein levels (Figure 6E and 6F; Figure IVB and VB) or Runx2 expression (Figure 6G), despite reducing mRNA levels of BMP4 (Figure 6H). Protein levels of pSMAD1/5/8 were increased after exogenous BMP4 treatment (Figure 6E and 6F), although Runx2 mRNA seemed unchanged (Figure 6G), and BMP4 expression was again reduced (Figure 6H). Similar changes were observed in murine MVICs (Figures VII, VIIIB, and XB in the Data Supplement).
Although total protein levels of β-catenin (Figure 6I; Figure IVC in the Data Supplement) were unaltered after exogenous TGF-β2, immunocytochemical analysis revealed increased nuclear translocation of β-catenin (Figure 6J; Figure VC in the Data Supplement). This coincided with increased mRNA levels of both the Wnt-target gene WISP1 (Figure 6K) and the ligand Wnt9A (Figure 6L). In contrast, BMP4 treatment did not affect levels or expression of Wnt-pathway proteins and genes, respectively (Figure 6I–6L). Similar changes were observed in murine MVICs (Figures VII, VIIIC, and XC in the Data Supplement).
Although protein levels of CD14 were unchanged (Figure 6M and 6N; Figure VD in the Data Supplement), in vitro treatment with TGF-β2 and BMP4 separately reduced mRNA levels of CD14 (Figure 6O), whereas TGF-β2 treatment induced a significant increase in CD83 expression in human MVICs (Figure 6P). Similar changes were observed in murine MVICs (Figure VII in the Data Supplement).
In Vivo Treatment With AngII Partially Recaptures Molecular and Phenotypic Features of Human MMVD in Murine Mitral Valves
The mean change in systolic blood pressure between baseline readings and day 14 postminipump implantation was an increase of 61±3 mm Hg versus an increase of 6±3 mm Hg in AngII and saline-infused mice, respectively (P<0.05). After 14 days of AngII treatment via minipumps, immunochemical staining with pSMAD2 was significantly increased (Figure 7A; Figures XIA and XIIA in the Data Supplement). Coincident with this, mRNA levels of fibrogenic genes, connective tissue growth factor and matrix metalloproteinase 2 (Figure 7B and 7C), and TGF-β2 ligand (Figure 7D) were increased after AngII infusion, whereas expression of the decoy receptor BAMBI was decreased (Figure 7E).
Although staining for pSMAD1/5/8 seemed unchanged in AngII versus saline infused mice (Figure 7F; Figures XIB and XIIB in the Data Supplement), expression of classically pro-osteogenic genes Msh homeobox 2 and Runx2 was significantly increased (Figure 7G and 7H), in the absence of altered expression of BMP4 and the intracellular BMP-pathway inhibitor transducer of erbB2 (Figure 7I and 7J).
Furthermore, AngII infusion was associated with increased staining for β-catenin in murine mitral valves (Figure 7K; Figures XIC and XIIC in the Data Supplement). This corresponded with increased expression of the Wnt target gene, WISP1 (Figure 7L) and decreased expression of the Wnt signaling inhibitor, Axin2 (Figure 7M), despite the absence of change in mRNA levels of Wnt9A ligand or the Wnt-receptor frizzled 8 (Figure 7N and 7O).
Although AngII infusion did not alter the expression of the cytokine interleukin-7 (Figure 7P) or pattern-recognition receptors CD14 and CD83 (Figure 7Q and 7R), it did induce increased mRNA levels of the immune receptor toll-like receptor 7 (Figure 7S). Furthermore, AngII infusion was associated with increased cellular proliferation in murine mitral valves, as evidenced by increased Ki-67 staining (Figures XIID, XIIE, XIID, and XIIE in the Data Supplement)
To exclude the possibility of myocardial contamination of mitral valve tissue, the cardiomyocyte-specific proteins MyH6 and Myl2 were measured in valve leaflets and ventricular tissue. Expression of both genes was dramatically higher in ventricular tissue than in leaflet tissue, and there were no differences between saline or AngII-treated groups within each tissue type (see Figure XIII in the Data Supplement ).
Finally, in the absence of a difference in ejection fraction (Figure 7T), LV mass (Figure 7U), LV end-systolic dimension (Figure XIVA in the Data Supplement) or LV end-diastolic dimension (Figure XIVB in the Data Supplement) between AngII and saline-infused mice, echocardiographic assessment on day 14 postminipump implantation revealed evidence of trace mitral regurgitation in ≈38% (8/21) of AngII-treated versus 17% (4/23) of saline-infused mice (Figure 7W; P=NS).
Using a nonbiased, high-throughput approach to molecular characterization, this study elucidates several novel molecular mechanisms that may underlie progression of MMVD to MVP. The key findings of our microarray analysis are (1) TGF-β signaling is increased in myxomatous mitral valve tissue because of increased ligand expression and derepression of canonical SMAD2/3 signaling, (2) canonical BMP and Wnt/β-catenin signaling pathways are increased in MMVD, (3) TGF-β, BMP, and Wnt/β-catenin pathways in MMVD are associated with matrix remodeling, procalcific and proproliferative cellular processes, and (4) activated immune cells are localized to myxomatous mitral valves in a manner independent of a classic inflammatory immune response. Our subsequent in vitro and in vivo experiments suggest that (1) upregulation of TGF-β signaling in MMVD may serve to transactivate Wnt/β-catenin signaling and (2) AngII signaling in vivo is sufficient to activate some, but not all, differentially activated signaling cascades observed in human MMVD.
TGF-β Signaling Is Derepressed in MMVD
A novel finding of this study is that induction of TGF-β signaling in MMVD is because of not only increased ligand expression but also increases in multiple molecules that facilitate transduction of canonical TGF-β signaling. Our finding that TGF-β2 is increased in MMVD is consistent with prior reports of increased TGF-β ligand expression in animal models of MVP and human MMVD.9–12 We also uncovered increased levels of the adaptor molecule Disabled2 (which has been shown to amplify canonical TGF-β signaling by recruiting receptor-activated SMADs2/3 to TGF-β receptor16), the transcriptional coactivator cyclic-AMP-responsive element-binding protein 5 and numerous TGF-β/SMAD2/3 target genes.
A major novel finding of this study, however, is that levels of the TGF-β decoy receptor, BAMBI,17 and the TGF-β signaling inhibitor SIK1,18 which may synergize with the inhibitory SMAD7, were reduced in MMVD. Furthermore, expression of the transcriptional corepressors c-Myc19 and TGF-β–induced factor 20 were also downregulated in MMVD. Importantly, although these molecular aberrations support current working models in which excessive TGF-β signaling may culminate in matrix remodeling and tissue fibrosis in MMVD, specifically they suggest that this phenomenon may in part be mediated by derepression of canonical signaling pathways.
Bone Morphogenetic Protein Signaling Is Activated in MMVD
Our current data also demonstrate robust activation of BMP signaling in MMVD. Although members of the BMP family are implicated in the pathogenesis of cardiovascular calcification,21 the role of BMP signaling in MVP, a phenotypically distinct disease entity not typically associated with leaflet calcification or aging, is yet to be clearly elucidated.
Consistent with 1 previous study,22 we found increases in BMP4 in myxomatous valves, and, to our knowledge, for the first time demonstrate that activation of this pathway is via canonical pSmad1/5/8 signaling. Similar to TGF-β signaling, BAMBI, SIK1, and transducer of erbB2, also negative regulators of canonical BMP signaling, were also reduced in myxomatous mitral valves. Critically, these data again support a novel disease model whereby derepression of TGFβ superfamily signaling is integral in the pathogenesis of MMVD.
Wnt/β-Catenin Signaling Is Activated in MMVD
To our knowledge, this study is the first to demonstrate activation of canonical Wnt signaling pathways in human MMVD. To be precise, expression of Wnt9A, Wnt-receptor frizzled 8 and classic Wnt-target genes (WISP1 and Runx2) were increased in myxomatous valves.23 Interestingly, we also uncovered increases in R-Spondin 2, which can increase Wnt signaling through attenuation of DKK1-dependent inhibition of Wnt ligands24,25
Although previous work described Wnt pathway–dependent induction of cellular proliferation in valvular interstitial cells in vitro,26 we think our data are the first to suggest that this phenomenon may be mediated by the Wnt ligand Wnt9A, and the Wnt-target gene Runx2. Recently, Runx2 has been shown to mediate cellular proliferation through repression of CDKN1A, mRNA levels of which are reduced in our microarray, and may thus play multiple, context-dependent roles in the development of MMVD.27–29 Although upregulation of Runx2 in myxomatous human mitral valves has been reported in a previous study, this has been predominantly discussed in the context of cell lineage determination (ie, promotion of chondrogenesis) and compared with the molecular changes seen in calcific aortic valve stenosis.30 Importantly, however, leaflet and annular calcification are not hallmarks of MMVD.
Abnormal Immune Infiltration in MMVD
To our knowledge, these data are the first to demonstrate robust evidence of immune cell infiltration in MMVD. In the present study, we found increases in the pattern recognition receptors CD14 and CD83 in myxomatous valves, suggestive of localization of immature and mature antigen presenting cells,31,32 respectively. We propose that future studies aimed at understanding interactions between interleukin-7, chemokine (C-X3-C motif) ligand 1, chemokine (C-X3-C motif) receptor 1, and immune cell infiltration may identify a maladaptive wound repair response in MMVD involving TGF-β, valvular interstitial cell activation, extracellular matrix remodeling, and functional leaflet prolapse.33,34
TGF-β2 and BMP4: Activation and Cross-Talk In Vitro
Through treatment of nonmyxomatous cultured MVICs with recombinant TGF-β2 and BMP4, we aimed to glean further insight into the relationship between and interplay among the various dysregulated signaling pathways identified by our microarray analysis.
Apart from increasing protein levels of pSMAD1/5/8, treatment of MVICs with exogenous BMP4 did not trigger robust increases in osteogenic gene expression, but did increase expression of negative regulators of BMP signaling (eg, BAMBI). Exogenous BMP4 also did not seem to transactivate canonical TGF-β or Wnt/β-catenin signaling cascades. Although this may be in part a function of treatment duration,22 our data collectively leave the precise contribution of BMP4 signaling in MMVD rather enigmatic.
As expected, in vitro TGF-β2 treatment induced canonical SMAD2 signaling and expression of multiple profibrotic genes. Our finding of increased expression of intracellular inhibitors of SMAD2/3 signaling (eg, SIK1, SMAD-specific E3 ubiquitin ligase 1, SMAD-specific E3 ubiquitin ligase 2) is in keeping with a negative feedback loop that would be expected after short-term TGF-β2 treatment. Interestingly, however, we found reduced expression of the decoy receptor BAMBI after TGF-β2 treatment, consistent with a paradoxical picture of a positive feedback loop. This finding suggests that TGF-β2–induced reductions in BAMBI in humans with MMVD may serve to further derepress and potentiate BMP and TGF-β signaling cascades.
Of equal significance is our finding that TGF-β2–treated MVICs demonstrate enhanced expression of Wnt9A and the classic Wnt-target gene WISP1. When considered in conjunction with the immunocytochemical evidence of increased nuclear β-catenin that suggests induction of canonical Wnt signaling—and despite unchanged total β-catenin—we propose that aberrant TGF-β activation may function to activate, or potentially transactivate, canonical Wnt/β-catenin signaling in MMVD. This is in keeping with prior evidence suggesting a key interaction between TGF-β and Wnt signaling pathways in promoting fibrosis in other tissues.35
Previous data have shown that TGF-β and BMP signaling are also capable of inducing transcription of Runx2, as well as promoting its activity as a transcription factor.36 Although our cell culture data do not conclusively support convergence of these signaling pathways on induction of Runx2 in MVICs, in vitro effects of TGF-β superfamily ligands are highly context-dependent (eg, concentration, duration, etc), and thus future studies are required to precisely characterize the contributions of TGF-β, BMP, and Wnt/β-catenin signaling to the transcriptional patterns observed in myxomatous valves.
TGF-β2 and BMP4: Interactions With Immune Signaling In Vitro
In the current study, treatment of human MVICS with either BMP4 or TGF-β2 resulted in significant reductions in the immature antigen presenting cell marker CD14. In contrast, treatment of cells with TGF-β2 resulted in small but significant increases in expression of CD83, a robust marker of mature antigen presenting cells. Although the functional consequences of these changes remain unclear, our understanding of the function of the immune system in the pathogenesis of MMVD is likely to be a critical future area of investigation.
AngII as a Common Link in MMVD?
Although hypertension is a long-standing risk factor for progression of MMVD to severe prolapse,14 AngII receptor activation has only recently been directly implicated in TGF-β–induced fibrogenic signaling in MVICs in vitro.11 In this study, treatment of mice with AngII induced robust activation of canonical SMAD-dependent TGF-β2 signaling and target gene expression. Although this observation is consistent with prior reports of AngII-mediated activation of TGF-β signaling in myocardial tissue,37 our work extends these findings by demonstrating that in vivo AngII can significantly reduce expression of the decoy receptor BAMBI in murine mitral valves. These findings broach the intriguing possibility that AngII-induced canonical TGF-β signaling may be potentiated by derepression at multiple points in this cascade.
Our observation that Wnt/β-catenin signaling is increased in AngII-infused mice is intriguing given emerging data that some of the transcriptional consequences of TGF-β receptor activation occur via activation (or transactivation) of canonical Wnt/β-catenin signaling. This is supported by our finding of increased expression of the Wnt-target gene WISP1 in AngII-treated mice. Critically, we also found that AngII-mediated increases in Runx2 are associated with upregulation of the cell proliferation marker Ki-67, which is consistent with our working hypothesis that Runx2 does not drive overt calcification in MMVD but instead promotes cellular proliferation. We propose that future investigation into interactions between TGF-β, Wnt/β-catenin, Runx2, and cell-cycle checkpoint proteins will lend key insights into the pathogenesis of MMVD.
Notably, AngII infusion did not recapture changes in BMP and Wnt ligands, or immune cell markers that we observed in human MMVD. AngII was similarly unable to induce a consistent and robust phenotype of prolapse and regurgitation in murine mitral valves. These findings suggest that 2 weeks of AngII-induced hypertension, at least in isolation, is insufficient to account for the entire fingerprint of human MMVD. Furthermore, this should incite caution before it is suggested that MMVD is simply an AngII-mediated disease or that monotherapy aimed at the renin angiotensin system would be a sufficient therapeutic strategy to slow progression of MMVD.11
Despite these drawbacks, we think that these data suggest that administration of AngII in mice may have some use in the evaluation and dissection of specific signaling cascades (eg, TGF-β and Wnt/β-catenin signaling) observed in MMVD using pharmacological interventions and genetically modified animals.
Complex Molecular Interactions in MMVD
Our microarray analyses provide evidence in support of the contribution of TGF-β, BMP, Wnt/β-catenin, and immune pathways in the pathogenesis of MMVD. However, the potential interactions and cross-talk between these pathways and their component genes/proteins are likely to be far more complex (Figure 5), and future experimental and interventional studies are required to more clearly elucidate the precise relationship between these signaling cascades. Importantly, understanding mechanisms mediating the recurrent and apparently widespread phenomenon of transcriptional derepression of pathological signaling cascades will be essential to the development of novel therapeutic interventions aimed at slowing progression of MMVD.
Study Limitations: Human Data
Several limitations of the current study warrant discussion. First, the limited amount of tissue available from human subjects meant it was not feasible to validate altered expression of all genes identified from microarray screens using RT-PCR. We therefore focused confirmatory analyses on key genes that would support evidence of canonical signaling pathway activation.
Second, although the human control valves obtained from transplant patients’ hearts were morphologically normal in appearance, they were likely exposed to undue stress and thus are not truly normal. Importantly, however, these valves were nonmyxomatous and historically have been deemed the best available controls for this line of investigation.
Although the majority of human valves studied were from men, cluster pattern analysis of high-throughput gene expression data did not identify any apparent differences between sexes. Furthermore, difficulty in acquiring age-matched samples of nonmyxomatous and myxomatous mitral tissue for study resulted in discrepant mean ages between the 2 groups. There were not, however, correlations between age and gene expression among any qRT-PCR–validated genes. Nevertheless, there are multiple plausible mechanisms that could contribute to differences in the biology and natural history of mitral valve disease with aging, across sexes, and when accounting for environmental stressors (eg, smoking), making these important areas for future investigation.
Statistically, a potential limitation of our primary statistical analysis was that we did not adjust for multiple comparisons to cast a broad net that would allow us to identify novel genes and pathways contributing to the development and progression of MMVD. After conducting a secondary, more stringent analysis of the data that adjusted for multiple comparisons (q<0.05), we found robust conservation of the vast majority of genes that were differentially regulated in our data set, and >90% of signaling cascades identified by ingenuity pathway analyses remained significantly differentially regulated between myxomatous and nonmyxomatous cohorts. Most importantly, TGF-β, BMP, Wnt/β-catenin, and immune signaling genes featured heavily and repeatedly in the most differentially regulated signaling cascades and did not change the interpretation of our results. Altogether, we think the presentation and focus on our primary analyses is in line with our goal of identifying a broad array of novel molecules and signaling cascades whose mechanistic role in MMVD can be interrogated using experimental models of MMVD in the future.
Finally, we acknowledge that our working model and proposed molecular contributors to MMVD have not been fully and mechanistically validated herein. Our cell culture experiments predominantly serve to highlight the exploratory and hypothesis-generating nature of the current article, and extensive in vitro studies are warranted to validate the roles of these pathways in MMVD.
Study Limitations: Mouse Data
One limitation of our in vivo animal studies that the molecular changes observed in AngII-treated animals may have been a consequence of hypertensive forces on the valve rather than AngII per se. Although our experimental design did not specifically address this, previous studies demonstrated that AngII is capable of driving deleterious molecular and phenotypic changes in cardiovascular tissues (including TGF-β expression, immune cell recruitment, fibroblast activation, etc) independent of changes in blood pressure.38–40 Future studies focused on understanding the interactions between AngII and blood pressure will provide critical insights into the pathogenesis of MMVD.
We also acknowledge that treatment of mice with AngII for 2 weeks represents a relatively short period of time. Although longer durations of treatment may augment the magnitude of molecular and phenotypic changes in mitral valves, such experiments are frequently associated with formation of aortic aneurysms or development of aortic valve regurgitation. Thus, future studies focused on treatment for longer durations will require development of protocols that do not confound data interpretation because of off target/confounding changes in cardiovascular function or attrition of animals from AngII-treated groups.
Collectively, these data provide evidence in support of a new disease framework in myxomatous mitral valve disease, whereby fibrogenic, osteogenic, and proliferative signaling are robustly activated in myxomatous valve tissue, and derepression of these signaling cascades may be a key permissive mechanism promoting disease progression. Although some of these changes appear reminiscent of the molecular signature present in calcific aortic valve disease, it is critical to note that the phenotypic consequences of these changes are dramatically different and are likely to act in a highly context-dependent manner. Furthermore, these signaling pathways may be subject to initiation and modulation by the immune system. Ultimately, we think that these findings open up numerous novel areas of research that may lead to nonsurgical therapies to slow progression of MMVD.
Sources of Funding
This work was supported by HL092235 (Dr Miller), HL111121 (Dr Miller), UL1TR000135 (Institutional Clinical and Translational Science Award), the Robert and Arlene Kogod Center on Aging at Mayo Clinic, and the Mayo Clinic Center for Regenerative Medicine.
The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.114.000921/-/DC1.
- Received April 21, 2014.
- Accepted March 12, 2015.
- © 2015 American Heart Association, Inc.
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Despite advances in the surgical management of myxomatous mitral valve degeneration (MMVD), biological mechanisms driving this disease are poorly understood, and nonsurgical therapeutic alternatives remain elusive. We used comprehensive whole genome expression arrays to elucidate novel molecular mechanisms underlying the development and progression of MMVD. In line with prior reports we identified increases in signaling of transforming growth factor-β, bone morphogenetic protein, and Wnt/β-catenin pathways in MMVD, and for the first time present evidence that reduced expression of endogenous inhibitors of these pathways may contribute to their overactivation. Although activation of these pathways seems to mirror molecular fingerprints described in aortic valve stenosis, we propose that pathways implicated in osteogenesis/calcification in the mitral valve act in a highly context-dependent manner, promoting matrix remodeling and proliferative processes that are characteristic of MMVD. Our data set also identifies novel markers of immune cell infiltration that seem to be ubiquitously elevated in myxomatous valves. Finally, we demonstrate that in vivo treatment of mice with angiotensin II recaptures only a subset of molecular changes observed in human disease and does not drive fulminant valvular prolapse, suggesting that angiotensin II blockade may not be effective as a monotherapy to slow MMVD. Collectively, we think that these data offer novel insights into the complex pathogenic basis of MMVD and provide a framework for future mechanistic experimentation into the causal relationships between these changes and development of clinically relevant valve disease, which will ultimately lead to the development of novel pharmacotherapies to slow or halt progression of MMVD.