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

Abstract

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 versican, the ADAMTS5 proteoglycan substrate, accumulates. ADAMTS5 deficient prevalvular mesenchyme also exhibited a reduction of α-smooth muscle actin and filamin A suggesting versican cleavage may be involved in TGFβ 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.

Spontaneous Aortic Regurgitation and Valvular Cardiomyopathy in Mice

“…Intact and cleaved versican levels remained elevated after treatment with pioglitazone in aortic valves from Wave mice. Expression of ADAMTS5, an enzyme which cleaves versican 11 , was decreased in Wave valves (Figure 4). Thus, increases in in tact versican and decreases in cleaved versican are consistent with increased levels of total proteoglycan in Wave valves, shown in Figure 1 J – L.…”

“…Intact versican, a proteoglycan that modulates properties of semilunar heart valves, 9,10 was increased in aortic valves from Wave mice at 6 months of age (Figure 4). Cleaved versican, a product of enzymatic breakdown of intact versican, was significantly reduced in aortic valves from Wave mice at 6 months of age (Figure 4).…”

“…Despite significant increases in polymeric intact versican, levels of cleaved versican are reduced in Wave mice at 6 months of age. Expression of ADAMTS5, an enzyme known to cleave versican, 11 was reduced in Wave mice. Expression of the gene encoding versican core protein was not increased, further supporting a post-transcriptional mechanism for proteoglycan excess in Wave valves.…”

Biosynthesis and Expression of a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats-15

“…Most notable are their roles during embryonic development where they mediate profound morphogenetic processes such as palatal shelf closure and pentameric digit formation (15,17). In addition, ADAMTS members are also required for normal heart development (18,19), and neural crest cell-derived pigmentation (16). In most cases, versican proteolysis by the ADAMTS proteoglycanases precedes normal development, suggesting this to be a key mediator of those processes.…”

“…Interdigital apoptosis is compromised in Adamts5, Adamts9, and Adamts20 combinatorial knock-out mice during embryogenesis, and an ADAMTS proteoglycanase generated versican (V1) cleavage product spanning from G1-DPEAAE 441 is required for BMP-mediated apoptosis to sculpt the developing autopod (17). Homozygous Adamts5 knock-out mice also present with abnormal heart valves (18,19). The absence of versican (V0/V1) processing at the defective site during embryonic development of respective Adamts proteoglycanase knock-out mice is a common precursor to each phenotype.…”