Adamts5-/- Mice Exhibit Altered Aggrecan Proteolytic Profiles That Correlate With Ascending Aortic Anomalies.

OBJECTIVE: Investigate the requirement of Aggrecan (Acan) cleavage during aortic wall development in a murine model with ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin-type motifs) 5 deficiency and bicuspid aortic valves. APPROACH: Mice with altered extracellular matrix remodeling of proteoglycans will be examined for anomalies in ascending aortic wall development. Neo-epitope antibodies that recognize ADAMTS cleaved Acan fragments will be used to investigate the mechanistic requirement of Acan turnover, in aortic wall development. RESULTS: Adamts5-/-;Smad2+/- mice exhibited a high penetrance of aortic anomalies (n=17/17); Adamts5-/-;Smad2+/- mice with bicuspid aortic valves (7/17) showed a higher number of anomalies than Adamts5-/-;Smad2+/- mice with tricuspid aortic valves. Single mutant Adamts5-/- mice also displayed a high penetrance of aortic anomalies (n=19/19) compared with wild type (n=1/11). Aortic anomalies correlated with Acan accumulation that was apparent at the onset of elastogenesis in Adamts5-/- mice. Neo-epitope antibodies that recognize the initial amino acids in the Acan cleaved fragments neo-FREEE, neo-GLGS, and neo-SSELE were increased in the Adamts5-/- aortas compared with WT. Conversely, neo-TEGE, which recognizes highly digested Acan core fragments, was reduced in Adamts5-/- mice. However, mice containing a mutation in the TEGE373↓374ALGSV site, rendering it noncleavable, had low penetrance of aortic anomalies (n=2/4). Acan neo-DIPEN and neo-FFGVG fragments were observed in the aortic adventitia; Acan neo-FFGVG was increased abnormally in the medial layer and overlapped with smooth muscle cell loss in Adamts5-/- aortas. CONCLUSIONS: Disruption of ADAMTS5 Acan cleavage during development correlates with ascending aortic anomalies. These data indicate that the mechanism of ADAMTS5 Acan cleavage may be critical for normal aortic wall development.

Intercalated cushion cells within the cardiac outflow tract are derived from the myocardial troponin T type 2 (Tnnt2) Cre lineage.

BACKGROUND: The origin of the intercalated cushions that develop into the anterior cusp of the pulmonary valve (PV) and the noncoronary cusp of the aortic valve (AV) is not well understood. RESULTS: Cre transgenes in combination with the Rosa TdTomato-EGFP reporter were used to generate three-dimensional lineage mapping of AV and PV cusps during intercalated cushion development. Tie2-Cre;EGFP was used to mark endothelial-derived mesenchymal cells, Wnt1-Cre;EGFP for cardiac neural crest and cardiac Troponin T (Tnnt2)Cre;EGFP, for myocardial lineage. The highest percentage of intercalated cushion cells at embryonic day (E) 12.5 was Tnnt2-Cre; EGFP positive; 68.0% for the PV and 50.0% AV. Neither Tnnt2 mRNA nor Tnnt2-Cre protein was expressed in the intercalated cushions; and the Tnnt2-Cre lineage intercalated cushion cells were also positive for the mesenchymal markers Sox9 and versican. Tnnt2-Cre lineage was present within the forming intercalated cushions from E11.5 and was present in the intercalated cushion derived PV and AV cusps and localized to the fibrosa layer at postnatal day 0. CONCLUSIONS: Intercalated cushions of the developing outflow tract are populated with Tnnt2-Cre derived cells, a Cre reporter previously used for tracing and excision of myocardial cells and not previously associated with mesenchymal cells. Developmental Dynamics 247:1005-1017, 2018. © 2018 Wiley Periodicals, Inc.

Development of myotendinous-like junctions that anchor cardiac valves requires fibromodulin and lumican.

BACKGROUND: There are many patients that exhibit connective tissue related cardiac malformations but do not have mutations in collagen genes. The Small Leucine Rich Proteoglycans (SLRP) fibromodulin (FMOD) and lumican (LUM) bind collagen and regulate fibril assembly in other biological contexts. RESULTS: FMOD deficient mice and double deficient FMOD; LUM mice exhibited anomalies in regions where cardiac valve tissue interdigitates with adjacent muscle for support. Ectopic connective and/or myocardial tissue(s) was associated with the more severe cardiac valve anomalies in FMOD; LUM deficient mice. At postnatal day 0 (P0) there was an increase in the mesenchymal cell number in the regions where valve cusps anchor in FMOD; LUM deficient mice compared to WT. The cardiac valve anomalies correlated with the highest levels of FMOD expression in the heart and also where myotendinous junctions (MTJ) components biglycan, collagen type I alpha 1, and collagen type VI, are also localized. CONCLUSIONS: The postnatal assembly of the collagen-rich ECM in regions where cardiac valves anchor, that we have designated 'myotendinous-like junctions' (MTLJ) requires the SLRPs FMOD and LUM. Moreover, FMOD and LUM may facilitate mesenchymal cell differentiation in late stages of cardiac valve development. Developmental Dynamics 245:1029-1042, 2016. © 2016 Wiley Periodicals, Inc.

Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly.

The ability of the heart to adapt to increased stress is dependent on the modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest that LUM deficient mice (lum(-/-)) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum(-/-) hearts have not been evaluated. These studies show that LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum(-/-) neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum(-/-) hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum(-/-) hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum(-/-) hearts. There was also a reduction in the ß and γ forms of collagenα1(I) in lum(-/-) hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum(-/-) hearts, indicating that LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart. Further investigation into the role of LUM may yield novel therapeutic targets and/or biomarkers for patients with cardiovascular disease.

Small leucine-rich proteoglycans exhibit unique spatiotemporal expression profiles during cardiac valve development.

BACKGROUND: Small Leucine Rich Proteoglycans (SLRPs) play a role in collagen fiber formation and also function as signaling molecules. Given the importance of collagen synthesis to the cardiovascular extracellular matrix (ECM), we examined the spatiotemporal expression of SLRPs, not previously investigated in the murine heart. RESULTS: Cardiac expression using antibodies specific for biglycan (BGN), decorin (DCN), fibromodulin (FMOD), and lumican (LUM) revealed distinct patterns among the SLRPs in mesenchymal-derived tissues. DCN showed the most intense localization within the developing valve cusps, while LUM was evident primarily in the hinge region of postnatal cardiac valves. BGN, DCN, and FMOD were immunolocalized to regions where cardiac valves anchor into adjacent tissues. Medial (BGN) and adventitial (BGN, DCN, FMOD and LUM) layers of the pulmonary and aortic arteries also showed intense staining of SLRPs but this spatiotemporal expression varied with developmental age. CONCLUSIONS: The unique expression patterns of SLRPs suggest they have adapted to specialized roles in the cardiovascular ECM. SLRP expression patterns overlap with areas where TGFß signaling is critical to the developing heart. Therefore, we speculate that SLRPs may not only be required to facilitate collagen fiber formation but may also regulate TGFß signaling in the murine heart.

Insufficient versican cleavage and Smad2 phosphorylation results in bicuspid aortic and pulmonary valves.

Bicuspid or bifoliate aortic valve (BAV) results in two rather than three cusps and occurs in 1-2% of the population placing them at higher risk of developing progressive aortic valve disease. Only NOTCH-1 has been linked to human BAV, and genetically modified mouse models of BAV are limited by low penetrance and additional malformations. Here we report that in the Adamts5(-/-) valves, collagen I, collagen III, and elastin were disrupted in the malformed hinge region that anchors the mature semilunar cusps and where the ADAMTS5 proteoglycan substrate versican, accumulates. ADAMTS5 deficient prevalvular mesenchyme also exhibited a reduction of α-smooth muscle actin and filamin A suggesting versican cleavage may be involved in TGFß signaling. Subsequent evaluation showed a significant decrease of pSmad2 in regions of prevalvular mesenchyme in Adamts5(-/-) valves. To test the hypothesis that ADAMTS5 versican cleavage is required, in part, to elicit Smad2 phosphorylation we further reduced Smad2 in Adamts5(-/-) mice through intergenetic cross. The Adamts5(-/-);Smad2(+/-) mice had highly penetrant BAV and bicuspid pulmonary valve (BPV) malformations as well as increased cusp and hinge size compared to the Adamts5(-/-) and control littermates. These studies demonstrate that semilunar cusp malformations (BAV and BPV) can arise from a failure to remodel the proteoglycan-rich provisional ECM. Specifically, faulty versican clearance due to ADAMTS5 deficiency blocks the initiation of pSmad2 signaling, which is required for excavation of endocardial cushions during aortic and pulmonary valve development. Further studies using the Adamts5(-/-); Smad2(+/-) mice with highly penetrant and isolated BAV, may lead to new pharmacological treatments for valve disease.

Increased Proteoglycanases in Pulmonary Valves after Birth Correlate with Extracellular Matrix Maturation and Valve Sculpting.

Increased mechanical forces on developing cardiac valves drive formation of the highly organized extracellular matrix (ECM) providing tissue integrity and promoting cell behavior and signaling. However, the ability to investigate the response of cardiac valve cells to increased mechanical forces is challenging and remains poorly understood. The developmental window from birth (P0) to postnatal day 7 (P7) when biomechanical forces on the pulmonary valve (PV) are altered due to the initiation of blood flow to the lungs was evaluated in this study. Grossly enlarged PV, in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from postnatal day 0 (P0) to P7; the Adamts5-/- aortic valves (AV) did not exhibit a phenotypic correction. We hypothesized that blood flow, initiated to the lungs at birth, alters mechanical load on the PV and promotes ECM maturation. In the Adamts5-/- PV, there was an increase in localization of the proteoglycan proteases ADAMTS1, MMP2, and MMP9 that correlated with reduced Versican (VCAN). At birth, Decorin (DCN), a Collagen I binding, small leucine-rich proteoglycan, exhibited complementary stratified localization to VCAN in the wild type at P0 but colocalized with VCAN in Adamts5-/- PV; concomitant with the phenotypic rescue at P7, the PVs in Adamts5-/- mice exhibited stratification of VCAN and DCN similar to wild type. This study indicates that increased mechanical forces on the PV at birth may activate ECM proteases to organize specialized ECM layers during cardiac valve maturation.

Altered versican cleavage in ADAMTS5 deficient mice; a novel etiology of myxomatous valve disease.

In fetal valve maturation the mechanisms by which the relatively homogeneous proteoglycan-rich extracellular matrix (ECM) of endocardial cushions is replaced by a specialized and stratified ECM found in mature valves are not understood. Therefore, we reasoned that uncovering proteases critical for 'remodeling' the proteoglycan rich (extracellular matrix) ECM may elucidate novel mechanisms of valve development. We have determined that mice deficient in ADAMTS5, (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) which we demonstrated is expressed predominantly by valvular endocardium during cardiac valve maturation, exhibited enlarged valves. ADAMTS5 deficient valves displayed a reduction in cleavage of its substrate versican, a critical cardiac proteoglycan. In vivo reduction of versican, in Adamts5(-/-) mice, achieved through Vcan heterozygosity, substantially rescued the valve anomalies. An increase in BMP2 immunolocalization, Sox9 expression and mesenchymal cell proliferation were observed in Adamts5(-/-) valve mesenchyme and correlated with expansion of the spongiosa (proteoglycan-rich) region in Adamts5(-/-) valve cusps. Furthermore, these data suggest that ECM remodeling via ADAMTS5 is required for endocardial to mesenchymal signaling in late fetal valve development. Although adult Adamts5(-/-) mice are viable they do not recover from developmental valve anomalies and have myxomatous cardiac valves with 100% penetrance. Since the accumulation of proteoglycans is a hallmark of myxomatous valve disease, based on these data we hypothesize that a lack of versican cleavage during fetal valve development may be a potential etiology of adult myxomatous valve disease.