The pro-atherogenic enzyme PAPP-A is active in eluates from human carotid and femoral atherosclerotic plaques.

Background: Pregnancy-associated plasma protein-A (PAPP-A) regulates bioavailability of insulin-like growth factor 1 (IGF1) in various tissues by proteolytic cleavage of a subset of IGF-binding proteins (IGFBPs). Pre-clinical studies have established a role of PAPP-A in atherosclerosis and proposed that targeting the proteolytic activity of PAPP-A has therapeutic value.This study aimed to investigate whether human atherosclerotic plaques contain proteolytically active PAPP-A, a prerequisite for further considering PAPP-A as a therapeutic target in patients. Methods: We obtained carotid (n = 9) and femoral (n = 11) atherosclerotic plaques from patients undergoing vascular surgery and incubated freshly harvested plaque tissue in culture media for 24 h. Subsequently, conditioned media were assayed for PAPP-A, STC2, IGFBP4, and IGF1 using immunoassays. Enzymatic activity of PAPP-A was assessed by its ability to process recombinant IGFBP4-IGF1 complexes - a specific substrate of PAPP-A - by Western blotting. Results: PAPP-A and STC2 were detectable in conditioned media from both carotid and femoral plaques, with higher STC2 concentrations in eluates from carotid plaque incubations (p = 0.02). IGFBP4 and IGF1 were undetectable. Conditioned media from all 20 plaques exhibited PAPP-A proteolytic activity. However, no correlation between PAPP-A concentration and its proteolytic activity was observed, whereas the PAPP-A: STC2 molar ratio correlated with PAPP-A activity (R2 = 0.25, p = 0.03). Conclusion: This study provides evidence for the presence of enzymatically active PAPP-A in atherosclerotic plaques and underscores the need for further investigating potential beneficial effects associated with targeting PAPP-A in atherosclerotic cardiovascular disease.

Local IGF Bioactivity Associates with High PAPP-A Activity in the Pericardial Cavity of Cardiovascular Disease Patients.

OBJECTIVE: Pregnancy-associated plasma protein-A (PAPP-A) has been suggested as a proatherogenic enzyme by its ability to locally increase insulin-like growth factor (IGF) activity through proteolytic cleavage of IGF binding protein-4 (IGFBP-4). Recently, stanniocalcin-2 (STC2) was discovered as an inhibitor of PAPP-A. This study aimed to investigate IGFBP-4, PAPP-A, and STC2 as local regulators of IGF bioactivity in the cardiac microenvironment by comparing levels in the pericardial fluid with those in the circulation of patients with cardiovascular disease. METHODS: Plasma and pericardial fluid were obtained from 39 patients undergoing elective cardiothoracic surgery, hereof 15 patients with type 2 diabetes. Concentrations of IGF-I, intact and fragmented IGFBP-4, PAPP-A, and STC2 were determined by immunoassays and IGF bioactivity by a cell-based assay. RESULTS: In pericardial fluid, the concentrations of total IGF-I, intact IGFBP-4, and STC2 were 72 ± 10%, 91 ± 5%, and 40 ± 24% lower than in plasma, while PAPP-A was 15 times more concentrated. The levels of the 2 IGFBP-4 fragments generated by PAPP-A and reflecting PAPP-A activity were elevated by more than 25%. IGF bioactivity was 62 ± 81% higher in the pericardial fluid than plasma. Moreover, pericardial fluid levels of both IGFBP-4 fragments correlated with the concentration of PAPP-A and with the bioactivity of IGF. All protein levels were similar in pericardial fluid from nondiabetic and diabetic subjects. CONCLUSIONS: PAPP-A increases IGF bioactivity by cleavage of IGFBP-4 in the pericardial cavity of cardiovascular disease patients. This study provides evidence for a distinct local activity of the IGF system, which may promote cardiac dysfunction and coronary atherosclerosis.

Pregnancy-associated plasma proteins and Stanniocalcin-2 - Novel players controlling IGF-I physiology.

IGF-I was originally discovered as a GH-dependent growth factor stimulating longitudinal growth. Currently, however, it has become evident that the biological activities of IGF-I extend well beyond those of a simple growth factor and impact such processes as insulin sensitivity, aging, cancer and cardiovascular disease. The vast majority of IGF-I is tightly bound to IGF-binding proteins (IGFBPs), which renders IGF-I unable to stimulate the IGF-I receptor (IGF-IR) in vivo. This binding means that liberation of IGF-I from the IGFBPs is an important step controlling IGF-I action. In this context, IGFBP-cleaving enzymes appear to play a key role. Enzymatic cleavage of the IGFBPs markedly lowers their ligand affinity, and as a consequence, IGF-I becomes liberated and hence available for stimulation of the IGF-IR. Two of the best-characterized IGFBP-cleaving enzymes are pregnancy-associated plasma protein-A (PAPP-A) and its paralog PAPP-A2. The two enzymes (often referred to as pappalysins) regulate the liberation of IGF-I in a highly controlled manner. PAPP-A is believed to act predominantly in tissues, serving to liberate IGF-I at the cell surface in close proximity to the IGF-IR. In keeping with this notion, mice lacking PAPP-A exhibit reduced body size, despite having normal circulating IGF-I concentrations. In contrast, human findings indicate that altered PAPP-A2 activity changes circulating IGF-I concentrations, although PAPP-A2 is also present in high concentrations in tissues. Thus, PAPP-A2 appears to impact circulating, as well as tissue, IGF-I activity. The enzymatic activity of PAPP-A and PAPP-A2 was recently discovered to be regulated by the protein Stanniocalcin-2 (STC2). By binding to the enzymatic sites of PAPP-A and PAPP-A2, STC2 inhibits their activity. To date, the majority of findings demonstrating the ability of pappalysins and STC2 to regulate IGF-I action are from preclinical studies. However, clinical studies are now beginning to emerge. In this review, we will summarize our data on STC2, PAPP-A and PAPP-A2 in humans. These results indicate that pappalysins and STC2 constitute an important IGF-I activity-regulating system that warrants further investigation.

The production and regulation of IGF and IGFBPs in human adipose tissue cultures.

BACKGROUND: Adipocytes express and secrete IGFs and IGFBPs; proteins with important effects on adipocyte homeostasis. However, the factors that control adipocyte generation of IGFs and IGFBPs are not clarified. AIM: To identify regulators of the synthesis of IGFs and IGFBs in adipose tissue. METHODS: Subcutaneous adipose tissue fragments (500 mg) from 7 healthy lean women were incubated for 48 h following addition of GH (50 µg/l), dexamethasone (DXM, 20 nM), insulin (100 nM), interleukin (IL)-1ß (50 ng/l), IL-6 (50 ng/l) and tumor-necrosis factor (TNF)-α (10 ng/l). Outcome parameters included tissue mRNA and culture media IGF and IGFBP levels. RESULTS: Adipose tissue cultures secreted more IGF-II than IGF-I protein (1.14±0.41 vs. 0.26±0.09 µg/l [mean±SEM]; P<0.02). IGF-I mRNA and protein levels were stimulated by GH (to 340% [153; 477] (median [interquartiles]) and 270±26%, respectively; P<0.003), and inhibited by IL-1ß (to 28% [21; 77] and 68±11%, respectively; P<0.003). TNF-α reduced IGF-I and IGF-II protein levels to 51±8% and 69±8%, respectively (P≤0.002), without affecting mRNA levels. IGF protein levels were unaffected by DXM, insulin and IL-6. All IGFBPs IGFBP-1 were expressed. IGFBP-4 was by far the most predominant IGFBP by immunoassay and WLB revealed two bands at 28 and 24 kDa, most likely representing glycosylated and non-glycosylated IGFBP-4. CONCLUSION: Adipose tissue cultures secrete more IGF-II than IGF-I, and predominantly IGFBP-4. The secretion of IGF-I is affected by GH, IL-1ß and TNF-α, whereas IGF-II is affected by TNF-α only. Hence, cytokines may control adipocyte homeostasis by affecting local IGF-generation.