Analysis of pregnancy-associated plasma protein A production in human adult cardiac progenitor cells

Abstract

IGF-binding proteins (IGFBPs) and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A) is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs) and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily, thus positively affecting IGF signalling. Therefore, our results strongly support the importance of PAPP-A for the IGFs/IGFBPs/PAPP-A axis in CPCs biology.

Figure 1

Quantification of PAPP-A secretion in CSps and CDCs conditioned media. PAPP-A concentrations were measured by immunofluorometric assay in individual 96-hour incubation CMs, randomly selected for CSps (n = 6) and CDCs (n = 5), and normalised to the total DNA content of the culture. PAPP-A normalised levels were significantly higher in CSp-CMs than in CDC-CMs (P = 0.018).

Figure 2

Immunoassayable PAPP-A using antibodies with different specificity. Titration of PAPP-A concentrations from CSp-CM (a) and CDC-CM (b) by three different EIAs. A0230 antibody (recognizing both dPAPP-A and PAPP-A/proMBP), 4PD4 antibody (specific for dPAPP-A), and 5H9 antibody (specific for the proMBP moiety) were used as capture antibodies for both CMs. In all cases, P0042 polyclonal antibody (HRP—conjugated anti-PAPP-A) was used for detection. The anti-proMBP/anti-PAPP-A antibody combination failed to recognize PAPP-A/proMBP complex in both CMs, whereas it was previously shown to be reactive with the pooled third trimester pregnancy serum-derived WHO 78/610 RP and with first and third trimester pregnancy human placental extracts [27].

Figure 3

Immunofluorescence and confocal analysis of PAPP-A in CSps and CDCs. Representative confocal images for the detection of PAPP-A in CSps (a–d) and CDCs (e-f) by immunofluorescence: PAPP-A/proMBP complex and free PAPP-A are stained by A0230; homodimeric PAPP-A is recognized by 4PD4 antibody and pro-MBP by 5H9. Note that (a–d) display different focal planes (core or surface) of the same CSp, showing how the spatial distribution of dPAPP-A and proMBP, respectively, varies inside the 3D structure of the CSp. A higher magnification panel of the corresponding evidenced area is shown as insert in (e).