Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves
Abstract:During embryogenesis the heart valves develop from undifferentiated mesenchymal endocardial cushions (EC), and activated interstitial cells of adult diseased valves share characteristics of embryonic valve progenitors. Twist1, a class II basic-helix-loop-helix (bHLH) transcription factor, is expressed during early EC development and is downregulated later during valve remodeling. The requirements for Twist1 down-regulation in the remodeling valves and the consequences of prolonged Twist1 activity were examined… Show more
“…We used a conditional Cre-induced transgene approach to constitutively express Twist1 in collagen II-expressing cells and their progeny. Activation of the transgene resulted in a modest (2.6 fold) increase in Twist1 transcripts over endogenous Twist1 expression, which was the same level as found using this transgene in other tissues (Chakraborty et al, 2010), yet the expression of the transgene was maintained as chondrocytes matured. Sustained expression of Twist1 in cartilage led to a growth phenotype, characterized by shortening of the limbs and reduced body mass.…”
Section: Discussionsupporting
confidence: 65%
“…1 decreased expression of Twist1 may be required for proper chondrocyte maturation. To test this, we used a Twist1 transgene that is conditionally activated by Cre recombinase (Chakraborty et al, 2010, Connerney et al, 2008, Connerney et al, 2006. Twist1 mice were crossed with Col2a1-Cre mice, which express Cre in proliferating chondrocytes, to activate constitutive Twist1 expression in these cells and their progeny.…”
Section: Impaired Skeletal Growth In Mice With Persistent Chondrocytementioning
Evidence from various in vitro gain and loss of function studies indicate that the bHLH transcription factor Twist1 negatively regulates chondrocyte differentiation; however limited information regarding Twist1 function in postnatal cartilage development and maintenance is available. Twist1 expression within the postnatal growth plate is restricted to immature, proliferating chondrocytes, and is significantly decreased or absent in hypertrophic chondrocytes. In order to examine the effect of maintaining the expression of Twist1 at later stages of chondocyte differentiation, we used type II collagen Cre (Col2-Cre) mice to activate a Cre-inducible Twist1 transgene specifically in chondrocytes (Col2-Twist1). At two weeks, postnatal growth was inhibited in Col2-Twist1 mice, as evidenced by limb shortening. Histological examination revealed abnormal growth plate structure, characterized by poor columnar organization of proliferating cartilaginous cells, decreased cellularity, and expansion of the hypertrophic zone. Moreover, structural defects within the growth plates of Col2-Twist1 transgenic mice included abnormal vascular invasion and focal regions of bony formation. Quantitative analysis of endochondral bone formation via micro-computed topography revealed impaired trabecular bone formation in the hindlimbs of Col2-Twist1 transgenic mice at various timepoints of postnatal development. Taken together, these findings indicate that regulated Twist1 expression contributes to growth plate organization and endochondral ossification to modulate postnatal longitudinal bone growth.
KEY WORDS: twist1, chondrocyte, growth plateTransition of chondrocytes from proliferation to terminal maturation within the growth plate is vital for longitudinal bone growth. Proliferating, immature chondrocytes synthesize an extracellular matrix (ECM) abundant in type II collagen and aggrecan. As cells differentiate, they mature into hypertrophic, postmitotic chondrocytes that enrich the ECM in type X collagen. Following hypertrophy, chondrocytes terminally mature and the calcified cartilage matrix is degraded by proteases and is infiltrated by blood vessels. Terminally mature chondrocytes then undergo apoptosis, thus facilitating the remodeling of the vascularized calcified matrix and its invasion by osteoblast precursors. Various families of transcription factors and signaling molecules, including transforming growth factor beta (TGF-b) and Wnts mediate this fine balance of chondrocyte proliferation, hypertrophy and terminal maturation necessary for longitudinal bone growth (Wuelling and Vortkamp, 2010). Further elucidation of the downstream effectors of these pathways may Int. J. Dev. Biol. 55: [641][642][643][644][645][646][647]
“…We used a conditional Cre-induced transgene approach to constitutively express Twist1 in collagen II-expressing cells and their progeny. Activation of the transgene resulted in a modest (2.6 fold) increase in Twist1 transcripts over endogenous Twist1 expression, which was the same level as found using this transgene in other tissues (Chakraborty et al, 2010), yet the expression of the transgene was maintained as chondrocytes matured. Sustained expression of Twist1 in cartilage led to a growth phenotype, characterized by shortening of the limbs and reduced body mass.…”
Section: Discussionsupporting
confidence: 65%
“…1 decreased expression of Twist1 may be required for proper chondrocyte maturation. To test this, we used a Twist1 transgene that is conditionally activated by Cre recombinase (Chakraborty et al, 2010, Connerney et al, 2008, Connerney et al, 2006. Twist1 mice were crossed with Col2a1-Cre mice, which express Cre in proliferating chondrocytes, to activate constitutive Twist1 expression in these cells and their progeny.…”
Section: Impaired Skeletal Growth In Mice With Persistent Chondrocytementioning
Evidence from various in vitro gain and loss of function studies indicate that the bHLH transcription factor Twist1 negatively regulates chondrocyte differentiation; however limited information regarding Twist1 function in postnatal cartilage development and maintenance is available. Twist1 expression within the postnatal growth plate is restricted to immature, proliferating chondrocytes, and is significantly decreased or absent in hypertrophic chondrocytes. In order to examine the effect of maintaining the expression of Twist1 at later stages of chondocyte differentiation, we used type II collagen Cre (Col2-Cre) mice to activate a Cre-inducible Twist1 transgene specifically in chondrocytes (Col2-Twist1). At two weeks, postnatal growth was inhibited in Col2-Twist1 mice, as evidenced by limb shortening. Histological examination revealed abnormal growth plate structure, characterized by poor columnar organization of proliferating cartilaginous cells, decreased cellularity, and expansion of the hypertrophic zone. Moreover, structural defects within the growth plates of Col2-Twist1 transgenic mice included abnormal vascular invasion and focal regions of bony formation. Quantitative analysis of endochondral bone formation via micro-computed topography revealed impaired trabecular bone formation in the hindlimbs of Col2-Twist1 transgenic mice at various timepoints of postnatal development. Taken together, these findings indicate that regulated Twist1 expression contributes to growth plate organization and endochondral ossification to modulate postnatal longitudinal bone growth.
KEY WORDS: twist1, chondrocyte, growth plateTransition of chondrocytes from proliferation to terminal maturation within the growth plate is vital for longitudinal bone growth. Proliferating, immature chondrocytes synthesize an extracellular matrix (ECM) abundant in type II collagen and aggrecan. As cells differentiate, they mature into hypertrophic, postmitotic chondrocytes that enrich the ECM in type X collagen. Following hypertrophy, chondrocytes terminally mature and the calcified cartilage matrix is degraded by proteases and is infiltrated by blood vessels. Terminally mature chondrocytes then undergo apoptosis, thus facilitating the remodeling of the vascularized calcified matrix and its invasion by osteoblast precursors. Various families of transcription factors and signaling molecules, including transforming growth factor beta (TGF-b) and Wnts mediate this fine balance of chondrocyte proliferation, hypertrophy and terminal maturation necessary for longitudinal bone growth (Wuelling and Vortkamp, 2010). Further elucidation of the downstream effectors of these pathways may Int. J. Dev. Biol. 55: [641][642][643][644][645][646][647]
“…TWIST1 is highly expressed during the formation of the cardiac cushion mesenchyme and plays important roles in valve mesenchyme proliferation and differentiation (Shelton and Yutzey, 2008;Chakraborty et al, 2010b). Owing to the key role of TWIST1 in heart valve development, we focused on characterizing the transcript levels of Twist1 in the Sox9 cKO valves.…”
Section: Sox9 Modulates a Core Network Of Tfs During Heart Valve Devementioning
confidence: 99%
“…In the absence of SOX9, Twist1 mRNA expression was reduced by approximately threefold in the valve mesenchyme. TWIST1 can induce proliferation and migration of valve mesenchyme during early valve formation (Shelton and Yutzey, 2008;Chakraborty et al, 2010b) and, following EMT, TWIST1 plays a role in regulating differentiation of the AVC mesenchyme (Vrljicak et al, 2012). When TWIST1 persists at later stages of valve development, it leads to increased mesenchyme proliferation, increased TBX20 expression, and primitive ECM (Chakraborty et al, 2010b).…”
Section: Sox9 Modulates the Transcript Levels Of Key Tfs In Heart Valmentioning
confidence: 99%
“…TWIST1 can induce proliferation and migration of valve mesenchyme during early valve formation (Shelton and Yutzey, 2008;Chakraborty et al, 2010b) and, following EMT, TWIST1 plays a role in regulating differentiation of the AVC mesenchyme (Vrljicak et al, 2012). When TWIST1 persists at later stages of valve development, it leads to increased mesenchyme proliferation, increased TBX20 expression, and primitive ECM (Chakraborty et al, 2010b). TWIST1 directly regulates Tbx20 (Lee and Yutzey, 2011), but, of note, Twist1-null hearts do not show a difference in Tbx20 levels in the AVC compared with WT (Vincentz et al, 2008).…”
Section: Sox9 Modulates the Transcript Levels Of Key Tfs In Heart Valmentioning
Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor SOX9 is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and valve formation. Despite this important role, little is known about how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in E12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Therefore, we compared regions of putative SOX9 DNA binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and context-independent SOX9-interacting regions throughout the genome. Analysis of contextindependent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissuespecific SOX9-interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom and Pitx2. Together, our data identify SOX9-coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation.
Tissue engineered heart valves (TEHV) could be useful in the repair of congenital or acquired valvular diseases due to their potential for growth and remodeling. The development of biomimetic scaffolds is a major challenge in heart valve tissue engineering. One of the most important structural characteristics of mature heart valve leaflets is their intrinsic anisotropy, which is derived from the microstructure of aligned collagen fibers in the extracellular matrix (ECM). In the present study, we used a directional electrospinning technique to fabricate fibrous poly-(glycerol sebacate):poly(caprolactone) (PGS:PCL) scaffolds containing aligned fibers, which resembled native heart valve leaflet ECM networks. In addition, the anisotropic mechanical characteristics of fabricated scaffolds were tuned by changing the ratio of PGS:PCL to mimic the native heart valve’s mechanical properties. Primary human valvular interstitial cells (VICs) attached and aligned along the anisotropic axes of all PGS:PCL scaffolds with various mechanical properties. The cells were also biochemically active in producing heart valve-associated collagen, vimentin, and smooth muscle actin as determined by gene expression. The fibrous PGS:PCL scaffolds seeded with human VICs mimicked the structure and mechanical properties of native valve leaflet tissues and would potentially be suitable for the replacement of heart valves in diverse patient populations.
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