Exploiting novel valve interstitial cell lines to study calcific aortic valve disease

Abstract

Calcific aortic valve disease (CAVD) involves progressive valve leaflet thickening and severe calcification, impairing leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to investigate CAVD mechanisms. However, to date, no published studies have utilised cell lines to investigate this process. The present study has therefore generated and evaluated the calcification potential of immortalized cell lines derived from sheep and rat VICs. Immortalised sheep (SAVIC) and rat (RAVIC) cell lines were produced by transduction with a recombinant lentivirus encoding the Simian virus (SV40) large and small T antigens (sheep), or large T antigen only (rat), which expressed markers of VICs (vimentin and α-smooth muscle actin). Calcification was induced in the presence of calcium (Ca; 2.7 mM) in SAVICs (1.9 fold; P<0.001) and RAVICs (4.6 fold; P<0.01). Furthermore, a synergistic effect of calcium and phosphate was observed (2.7 mM Ca/2.0 mM Pi) on VIC calcification in the two cell lines (P<0.001). Analysis of SAVICs revealed significant increases in the mRNA expression of two key genes associated with vascular calcification in cells cultured under calcifying conditions, runt related transcription factor-2 (RUNX2;1.3 fold; P<0.05 in 4.5 mM Ca) and sodium-dependent phosphate transporter-1 (PiT1; 1.2 fold; P<0.05 in 5.4 mM Ca). A concomitant decrease in the expression of the calcification inhibitor matrix Gla protein (MGP) was noted at 3.6 mM Ca (1.3 fold; P<0.01). Assessment of RAVICs revealed alterations in Runx2, Pit1 and Mgp mRNA expression levels (P<0.01). Furthermore, a significant reduction in calcification was observed in SAVICs following treatment with established calcification inhibitors, pyrophosphate (1.8 fold; P<0.01) and etidronate (3.2 fold; P<0.01). Overall, the present study demonstrated that the use of immortalised sheep and rat VIC cell lines is a convenient and cost effective system to investigate CAVD in vitro, and will make a useful contribution to increasing current understanding of the pathophysiological process.

The genetic consequences of dog breed formation—Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels

“…In line with this, ENCODE data on SCREEN ( https://screen.wenglab.org ) predict that the homologous positions of NEBL 1 and 4 in the human genome are located in cis-regulatory elements (Table 25 in S1 Text ) [ 46 ]. We next used electrophoretic mobility shift assays (EMSA) to investigate potential protein binding to NEBL 1-, 2-, 3- and 4 in-vitro , using nuclear cell extracts from three different cell lines: i) RAVIC (a model for heart valve interstitial cell biology [ 47 ]); ii) H9C2 (differentiated into cardiomyocytes); and iii) MDCK (derived from dog kidney cells and used to finetune the protocol using probe and protein that both originate from dog). For NEBL 3, we documented no clear binding for either allele in any of the cell lines tested (Fig 10 in S1 Text ).…”

“…Protein binding to all oligos was assayed in nuclear extracts from three different cell types; i) the MDCK cell line is derived from dog (Cocker spaniel) kidney tubule epithelia and was used as a representative of a non-cardiac derived cell line that allowed us to test binding in a setting where both protein and probe sequence originate from dog; ii) In absence of immortalized cell lines derived from mitral valve, we used RAVIC cells as a model for heart valve interstitial cell biology. RAVIC cells derive from rat (Sprague Dawley rats) aortic valvular interstitial cells [ 47 ]. Similar to the mitral valve, the aortic valve is located in the left side of the heart.…”

Scanning electron microscopy for blood micro-crystals in aortic stenosis patients

“…It is known that increasing levels of calcium and phosphate, within the physiological range, induce calcification of valvular interstitial cells in vitro [ 12 ]. Serum is a metastable solution with respect to calcium phosphate precipitation.…”

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

“…First, the size of VIC population within rat valves is very small, and therefore many animals are needed in order to generate sufficient cell numbers for extensive in vitro studies. However, it is possible to overcome this limitation by undertaking preliminary studies in immortalized valve interstitial cell lines, as recently reported by us 18 , and subsequently employing primary cultures to verify and extend these findings.…”