TLR4 Stimulation Promotes Human AVIC Fibrogenic Activity through Upregulation of Neurotrophin 3 Production

Abstract

Background: Calcific aortic valve disease (CAVD) is a chronic inflammatory disease that manifests as progressive valvular fibrosis and calcification. An inflammatory milieu in valvular tissue promotes fibrosis and calcification. Aortic valve interstitial cell (AVIC) proliferation and the over-production of the extracellular matrix (ECM) proteins contribute to valvular thickening. However, the mechanism underlying elevated AVIC fibrogenic activity remains unclear. Recently, we observed that AVICs from diseased aortic valves express higher levels of neurotrophin 3 (NT3) and that NT3 exerts pro-osteogenic and pro-fibrogenic effects on human AVICs. Hypothesis: Pro-inflammatory stimuli upregulate NT3 production in AVICs to promote fibrogenic activity in human aortic valves. Methods and Results: AVICs were isolated from normal human aortic valves and were treated with lipopolysaccharide (LPS, 0.20 µg/mL). LPS induced TLR4-dependent NT3 production. This effect of LPS was abolished by inhibition of the Akt and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways. The stimulation of TLR4 in human AVICs with LPS resulted in a greater proliferation rate and an upregulated production of matrix metallopeptidases-9 (MMP-9) and collagen III, as well as augmented collagen deposition. Recombinant NT3 promoted AVIC proliferation in a tropomyosin receptor kinase (Trk)-dependent fashion. The neutralization of NT3 or the inhibition of Trk suppressed LPS-induced AVIC fibrogenic activity. Conclusions: The stimulation of TLR4 in human AVICs upregulates NT3 expression and promotes cell proliferation and collagen deposition. The NT3-Trk cascade plays a critical role in the TLR4-mediated elevation of fibrogenic activity in human AVICs. Upregulated NT3 production by endogenous TLR4 activators may contribute to aortic valve fibrosis associated with CAVD progression.

Upregulation of Cartilage Oligomeric Matrix Protein and Bone Morphogenetic Protein-2 May Associate with Calcific Aortic Valve Disease

“…[20] Additionally, BMP2 is a marker of vascular cells' osteogenic phenotypic change, which is a critical mechanistic step in the development of CAVD. [21] Kim et al [18] found that COMP can directly bind to the C-terminus of BMP2, as confirmed by Co-IP and GST pull-down assays. Consistently, we found that COMP and BMP2 were co-located in CAVD group valves in this study.…”

“…BMP2 and transforming growth factor-β (TGF-β) can mediate biglycan-induced pro-osteogenic reprogramming in aortic valve interstitial cells [20] . Additionally, BMP2 is a marker of vascular cells’ osteogenic phenotypic change, which is a critical mechanistic step in the development of CAVD [21] . Kim et al [18] found that COMP can directly bind to the C-terminus of BMP2, as confirmed by Co-IP and GST pull-down assays.…”

Dissecting Calcific Aortic Valve Disease—The Role, Etiology, and Drivers of Valvular Fibrosis

“…As mentioned before, TLRs and neutrophin 3 are involved in myofibroblast activation (118). Neutrophin neutralization using specific antibodies or inhibition of the downstream signaling was shown to inhibit the activation in vitro; whether a clinical application is thinkable needs to be evaluated.…”

“…Stimulation of TLR4 with lipopolysaccharide activates the Akt and ERK1/2 pathway and increases the expression of neurotrophin 3. This activation subsequently results in VIC proliferation and increased collagen 3 and MMP9 production with implications for pathological ECM remodeling ( 118 ).…”

Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

“…Moreover, we observed that human AVICs express functional Toll-like receptors (TLRs), and AVICs isolated from diseased aortic valves have higher levels of TLR2 and TLR4 [10]. More importantly, stimulation of TLRs in human AVICs not only induces inflammatory responses [7][8][9]11], but also elevates cellular osteogenic and fibrogenic activities [8][9][10][12][13][14], and the pro-inflammatory and pro-osteogenic pathways in human AVICs interact to modulate cellular inflammatory and osteogenic activities [15]. Thus, TLRs may play a mechanistic role in mediating CAVD progression [16].…”