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Alzheimer's disease (AD) is a progressive neurodegenerative disease of age with no effective preventative or treatment approaches. Deeper understanding of the mechanisms underlying the accumulation of toxic β-amyloid oligopeptides and the formation of amyloid plaque in AD has the potential to identify new therapeutic targets. Prior research links the insulin-like growth factor (IGF) system to pathologic mechanisms underlying AD. Suppression of local IGF-I receptor (IGFIR) signaling in AD mice has been shown to reduce plaque formation in the brain and delay neurodegeneration and behavioral changes. However, direct inhibitors of IGFIR signaling are not a viable treatment option for AD due to the essentiality of the IGFIR in physiological growth and metabolism. We have previously demonstrated a more selective means to reduce local IGFIR signaling through inhibition of PAPP-A, a novel zinc metalloprotease that regulates local IGF-I bioavailability through cleavage of inhibitory IGF binding proteins. Here we tested if deletion of PAPP-A in a mouse model of AD provides protection against pathology and behavioral changes. We show that compared to AD mice, AD/PAPP-A KO mice had significantly less plaque burden, reduced astrocytic activation, decreased IGF-IR activity, and improved cognition. Human senile AD plaques showed specific immunostaining for PAPP-A. Thus, inhibition of PAPP-A expression or activity may represent a novel treatment strategy for AD., Competing Interests: Declaration of competing interest DJB has a potential conflict related to his research. He is co-inventor on patents held by Mayo Clinic, patent applications licensed or to be filed by Unity Biotechnology and is a Unity Biotechnology shareholder. Research in the Baker laboratory has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo conflict of interest policies. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Proptosis in Thyroid Eye Disease (TED) can result in facial disfigurement and visual dysfunction. Treatment with Insulin-like growth factor I receptor (IGF-IR) inhibitors has been shown to be effective in reducing proptosis but with side effects., Objective: To test the hypothesis that inhibition of IGF-IR indirectly and more selectively with PAPP-A inhibitors attenuates IGF-IR signaling in TED., Design: Informed consent was obtained from TED patients undergoing surgery, and retro-orbital tissue collected for fibroblast isolation and culture., Setting: Surgeries were performed in Mayo Clinic operating suites. Cell culture was performed in a sterile tissue culture facility., Patient Samples: Retro-orbital tissue was collected from 19 TED patients., Interventions: Treatment of TED fibroblasts with pro-inflammatory cytokines. Flow separation of CD34- and CD34+ orbital fibroblasts, the latter representing infiltrating fibrocytes into the orbit in TED., Main Outcome Measures: PAPP-A expression and proteolytic activity, IGF-I stimulation of phosphatidylinositol 3 kinase/Akt pathway and inhibition by immuno-neutralizing antibodies against PAPP-A, CD34+ status and associated PAPP-A and IGF-IR expression., Results: Pro-inflammatory cytokines markedly increased PAPP-A expression in TED fibroblasts. IGF-IR expression was not affected by cytokine treatment. Inhibition of PAPP-A's proteolytic activity suppressed IGF-IR activation in orbital fibroblasts from TED patients. TED fibroblasts that were CD34+ represented ∼80% of the cells in culture and accounted for ∼70% of PAPP-A and IGF-IR expressing cells., Conclusions: These results support a role for PAPP-A in TED pathogenesis and indicate the potential for novel therapeutic targeting of the IGF axis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Pregnancy-associated plasma protein-A (PAPP-A) was first identified in the early 1970s as a placental protein of unknown function, present at high concentrations in the circulation of pregnant women. In the mid-to-late 1990s, PAPP-A was discovered to be a metzincin metalloproteinase, expressed by many nonplacental cells, that regulates local insulin-like growth factor (IGF) activity through cleavage of high-affinity IGF binding proteins (IGFBPs), in particular IGFBP-4. With PAPP-A as a cell surface-associated enzyme, the reduced affinity of the cleavage fragments results in increased IGF available to bind and activate IGF receptors in the pericellular environment. This proteolytic regulation of IGF activity is important, since the IGFs promote proliferation, differentiation, migration, and survival in various normal and cancer cells. Thus, there has been a steady growth in investigation of PAPP-A structure and function outside of pregnancy. This review provides historical perspective on the discovery of PAPP-A and its structure and cellular function, highlights key studies of the first 50 years in PAPP-A research, and introduces new findings from recent years., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The pappalysin metalloproteinases, PAPP-A and PAPP-A2, have emerged as highly specific proteolytic enzymes involved in the regulation of insulin-like growth factor (IGF) signaling. The only known pappalysin substrates are a subset of the IGF binding proteins (IGFBPs), which bind IGF-I or IGF-II with high affinity to antagonize receptor binding. Thus, by cleaving IGFBPs, the pappalysins have the potential to increase IGF bioactivity and hence promote IGF signaling. This is relevant both in systemic and local IGF regulation, in normal and several pathophysiological conditions. Stanniocalcin-1 and -2 were recently found to be potent pappalysin inhibitors, thus comprising the missing components of a complete proteolytic system, the stanniocalcin-PAPP-A-IGFBP-IGF axis. Here, we provide the biological context necessary for understanding the properties of this molecular network, and we review biochemical data, animal experiments, clinical data, and genetic data supporting the physiological operation of this branch as an important part of the IGF system. However, although in vivo data clearly illustrate its power, it is a challenge to understand its subtle operation, for example, multiple equilibria and inhibitory kinetics may determine how, where, and when the IGF receptor is stimulated. In addition, literally all of the regulatory proteins have suspected or known activities that are not directly related to IGF signaling. How such activities may integrate with IGF signaling is also important to address in the future., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Senescence is a cellular response to various stressors characterized by irreversible cell cycle arrest, resistance to apoptosis and expression of a senescence-associated secretory phenotype (SASP). Interestingly, studies where senescent cells were deleted in mice produced beneficial effects similar to those where the zinc metalloproteinase, PAPP-A, was deleted in mice. In this study, we investigated the effect of senescence on PAPP-A secretion and activity in primary cultures of adult human pre-adipocytes. Cultured pre-adipocytes were isolated from subcutaneous (Sub) and omental (Om) fat. Senescence was induced with low dose etoposide. PAPP-A protein was measured by an ultrasensitive PAPP-A ELISA. PAPP-A proteolytic activity was measured by a specific substrate cleavage assay. Senescence significantly increased PAPP-A levels in both Sub and Om conditioned medium (CM) 8- to 15-fold over non-senescent CM. Proteolytic activity reflected PAPP-A protein with 12- to 18-fold greater activity in senescent CM versus non-senescent CM. Furthermore, PAPP-A was found at high levels on the surface of extracellular vesicles secreted by senescent pre-adipocytes and was proteolytically active. In conclusion, we identified enzymatically active PAPP-A as a component of human pre-adipocyte SASP. This recognition warrants further investigation of PAPP-A as a new biomarker for senescence and a potential therapeutic target to control of the spread of senescence in adipose tissue., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Brown bears conserve muscle and bone mass during 6 mo of inactive hibernation. The molecular mechanisms underlying hibernation physiology may have translational relevance for human therapeutics. We hypothesize that protective mechanisms involve increased tissue availability of insulin-like growth factors (IGFs). In subadult Scandinavian brown bears, we observed that mean plasma IGF-1 and IGF-2 levels during hibernation were reduced to 36 ± 10% and 56 ± 15%, respectively, compared with the active state ( n = 12). Western ligand blotting identified IGF-binding protein (IGFBP)-3 as the major IGFBP in the active state, whereas IGFBP-2 was codominant during hibernation. Acid labile subunit (ALS) levels in hibernation were reduced to 41±16% compared with the active state ( n = 6). Analysis of available grizzly bear RNA sequencing data revealed unaltered liver mRNA IGF-1 , IGFBP-2 , and IGFBP-3 levels, whereas ALS levels were significantly reduced during hibernation ( n = 6). Reduced ALS synthesis and circulating levels during hibernation should prompt a shift from ternary IGF/IGFBP/ALS to smaller binary IGF/IGFBP complexes, thereby increasing IGF tissue availability. Indeed, size-exclusion chromatography of bear plasma demonstrated a shift to lower molecular weight IGF-containing complexes in the hibernating versus the active state. Furthermore, we note that the major IGF-2 mRNA isoform expressed in livers in both Scandinavian brown bears and grizzly bears was an alternative splice variant in which Ser29 is replaced with a tetrapeptide possessing a positively charged Arg residue. Homology modeling of the bear IGF-2/IGFBP-2 complex showed the tetrapeptide in proximity to the heparin-binding domain involved in bone-specific targeting of this complex. In conclusion, this study provides data which suggest that increased IGF tissue availability combined with tissue-specific targeting contribute to tissue preservation in hibernating bears. NEW & NOTEWORTHY Brown bears shift from circulating ternary IGF/IGFBP/ALS complexes in the active state to binary IGF/IGFBP complexes during hibernation, indicating increased tissue IGF-bioactivity. Furthermore, brown bears use a splice variant of IGF-2, suggesting increased bone-specific targeting of IGF anabolic signaling.

Background: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions., Methods and Results: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2., Conclusion: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with few effective treatment options. We found a highly significant correlation between pregnancy-associated plasma protein (PAPP)-A expression in IPF lung tissue and disease severity as measured by various pulmonary and physical function tests. PAPP-A is a metalloproteinase that enhances local insulin-like growth factor (IGF) activity. We used primary cultures of normal adult human lung fibroblasts (NHLF) to test the hypothesis that PAPP-A plays an important role in the development of pulmonary fibrosis. Treatment of NHLF with pro-fibrotic transforming growth factor (TGF)-β stimulated marked increases in IGF-I mRNA expression (>20-fold) and measurable IGF-I levels in 72-h conditioned medium (CM). TGF-β treatment also increased PAPP-A levels in CM fourfold (p = 0.004) and proteolytic activity ~2-fold. There was an indirect effect of TGF-β to stimulate signaling through the PI3K/Akt pathway, which was significantly inhibited by both IGF-I-inactivating and PAPP-A inhibitory antibodies. Induction of senescence in NHLF increased PAPP-A levels in CM 10-fold (p = 0.006) with attendant increased proteolytic activity. Thus, PAPP-A is a novel component of the senescent lung fibroblast secretome. In addition, NHLF secreted extracellular vehicles (EVs) with surface-bound active PAPP-A that were increased fivefold with senescence. Regulation of PAPP-A and IGF signaling by TGF-β and cell senescence suggests an interactive cellular mechanism underlying the resistance to apoptosis and the progression of fibrosis in IPF. Furthermore, PAPP-A-associated EVs may be a means of pro-fibrotic, pro-senescent communication with other cells in the lung and, thus, a potential therapeutic target for IPF., (© 2022 Wiley Periodicals LLC.)

PAPP-A knock-out (KO) mice are a valuable model for investigating the effects of down-regulating localized insulin-like growth factor (IGF) action, which has been shown to extend lifespan and healthspan when the PAPP-A gene is globally deleted. Based on previous mouse models of brain-specific reduction in IGF signaling associated with longevity, we sought to generate brain-specific PAPP-A KO mice and determine effects on metabolism and lifespan. Mice with the PAPP-A gene floxed (fPAPP-A) were crossed with Nestin promoter-driven Cre recombinase transgenic mice. This cross-breeding of mice for Nestin-Cre and mice with other floxed target alleles has been used extensively to investigate brain-specific effects. Our cross-breeding generated four genotypes for study: fPAPP-A/Nestin positive (brain-specific PAPP-A KO); fPAPP-A/Nestin negative (Control for floxed PAPP-A); WT/Nestin positive (Control for Nestin-Cre); WT/Nestin negative (Wild-type Control). The basic genotype screen of neonatal tail snip DNA clearly indicated PAPP-A gene status and the presence (pos) or absence (neg) of Nestin-Cre. We then determined tissue specificity of PAPP-A gene excision. We had expected fPAPP-A/pos mice to be relatively brain-specific for PAPP-A gene deletion and the controls (fPAPP-A/neg, WT/neg and WT/pos mice) to show no effect on PAPP-A expression in brain or other tissues. However, in fPAPP-A/neg mice we found evidence of PAPP-A excision in all tissues examined, i.e., in the presumed absence of Nestin-Cre, indicating germline recombination. We further found that fPAPP-A/pos mice showed near complete excision of the PAPP-A gene in brain, but some also showed germline recombination affecting all tissues tested. To determine if the level of excision indicated by tissue genotyping approximated PAPP-A mRNA expression, we performed RT-qPCR. fPAPP-A/pos mice that showed markedly decreased whole brain PAPP-A mRNA expression (~80%), with little or no effect on expression in the other tissues tested, were designated as "brain-specific" PAPP-A KO. fPAPP-A/pos mice that showed germline recombination had similar decreases in PAPP-A expression in brain but also showed 40-65% decreased PAPP-A mRNA expression in other tissues as well, which was especially striking in kidney, tibia, thymus and spleen. These were designated as "non-specific" PAPP-A KO mice. With unknown and unpredictable specificity until harvest, we chose to assess a surrogate marker of lifespan i.e., thymic involution, in 15- to 18-month-old fPAPP-A/pos and WT/pos mice, the latter an important control for a possible effect of Nestin-Cre per se. Diminished thymic involution as indicated by increased thymic weight (135%, P = 0.035) and decreased histological disruption was seen in "non-specific" PAPP-A KO mice, similar to what was previously reported in 18-month-old global PAPP-A KO mice. There was no significant difference between "brain-specific" PAPP-A KO and control mice. This study highlights the importance of thorough characterization of assumed tissue-specific mouse models and awareness of potential germline recombination for proper data interpretation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pathogenicity of visceral adipose tissue (VAT) has been linked to the metabolic stress of enlarging mature adipocytes and a limited ability to recruit new adipocytes. One of the major distinguishing features of VAT preadipocytes is the high expression of the zinc metalloprotease, pregnancy-associated plasma protein-A (PAPP-A), when compared to subcutaneous adipose tissue (SAT). In this study we used 2 different approaches to investigate the effect of PAPP-A inhibition on different fat depots in mice on a high-fat diet (HFD) for 15 weeks. Conditional knockdown of PAPP-A gene expression in female adult mice resulted in significant decreases of 30% to 40% in adipocyte size in VAT (mesenteric and pericardial depots) compared to control mice. There was no effect on SAT (inguinal) or intra-abdominal perigonadal fat. Liver lipid was also significantly decreased without any effect on heart and skeletal muscle lipid. We found similar effects when using a pharmacological approach. Weekly injections of a specific immunoneutralizing monoclonal antibody (mAb-PA 1/41) or isotype control were given to male and female wild-type mice on HFD for 15 weeks. Adipocyte size was significantly decreased (30%-50%) only in VAT with mAb-PA 1/41 treatment. In this model, cell number was significantly increased in mesenteric fat in mice treated with mAb-PA 1/41, suggesting hyperplasia along with reduced hypertrophy in this VAT depot. Gene expression data indicated a significant decrease in F4/80 (macrophage marker) and interleukin-6 (proinflammatory cytokine) and a significant increase in adiponectin (anti-inflammatory adipokine with beneficial metabolic effects) in mesenteric fat compared to inguinal fat in mice treated with mAb-PA 1/41. Furthermore, there was significantly decreased liver lipid content with mAb-PA 1/41 treatment. Thus, using 2 different models systems we provide proof of principle that PAPP-A inhibition is a potential therapeutic target to prevent visceral obesity and its metabolic sequelae, such as fatty liver., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Suppression of the insulin-like growth factor-1 (IGF-1) signaling pathway reduces age-related disorders and increases lifespan across species, making the IGF-1 pathway a key regulator of aging. Previous in vitro intervertebral disc cell studies have reported the pro-anabolic effect of exogenously adding IGF-1 on matrix production. However, the overall effects of suppressing IGF-1 signaling on age-related intervertebral disc degeneration (IDD) is not known. Here, the effects of suppressing IGF-1 signaling on age-related IDD in vivo were examined using PAPPA -/- mice. These are animals with targeted deletion of pregnancy-associated plasma protein A (PAPPA), the major protease that cleaves inhibitory IGF binding proteins that control bioavailability of IGF-1 for cell signaling. Compared to age-matched wild-type (Wt) littermates, reduced levels of matrix proteoglycan (PG) and aggrecan were seen in discs of 23-month old PAPPA -/- mice. Decreased aggrecanolysis and expression of two key catabolic markers, matrix metalloproteinase-3 and a disintegrin and metalloproteinase with thrombospondin motifs-4, were also observed in discs of old PAPPA -/- mice compared to Wt littermates. Suppressing IGF-1 signaling has been implicated to shift cellular metabolism toward maintenance rather than growth and decreasing cellular senescence. Along this line, discs of old PAPPA -/- mice also exhibited lower cellular senescence, assessed by p53 and lamin B1 markers. Collectively, the data reveal complex regulation of disc matrix homeostasis by PAPPA/IGF-1 signaling during chronologic aging, that is, reduced IGF-1 bioavailability confers the benefit of decreasing disc cellular senescence and matrix catabolism but also the disadvantage of decreasing disc PG matrix anabolism. This pathway requires further mechanistic elucidation before IGF-1 could be considered as a therapeutic growth factor for treating IDD., Competing Interests: The authors declare no potential conflict of interest., (© 2020 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of Pkd1RC/RC mice, a model of ADPKD. Pregnancy-associated plasma protein A (PAPP-A), a metalloproteinase that cleaves inhibitory IGF binding proteins (IGFBPs), increasing the local bioactivity of IGF-1, was highly induced in the kidney of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed that PAPP-A transcription in ADPKD was mainly regulated through the cAMP/CREB/CBP/p300 pathway. Pappa deficiency effectively inhibited the development of cysts in the Pkd1RC/RC mice. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with a monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1RC/RC mice and ex vivo in embryonic kidneys. These data indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a therapeutic strategy for ADPKD that involves the inhibition of PAPP-A.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase with a well-established role in releasing bioactive insulin-like growth factor-1 (IGF-1) from IGF-binding protein-2, -4, and -5 by proteolytic processing of these. The IGF system has repeatedly been suggested to be involved in the pathology of atherosclerosis, and both PAPP-A and IGF-1 are proposed biomarkers and therapeutic targets for this disease. Several experimental approaches based on atherosclerosis mouse models have been undertaken to obtain causative and mechanistic insight to the role of these molecules in atherogenesis. However, reports seem conflicting. The literature suggests that PAPP-A is detrimental, while IGF-1 is beneficial. This raises important questions that need to be addressed. Here we summarize the various studies and discuss potential underlying explanations for this seemingly inconsistency with the objective of better understanding complexities and limitations when manipulating the IGF system in mouse models of atherosclerosis. A debate clarifying what's up and what's down is highly warranted going forward with the ultimate goal of improving atherosclerosis therapy by targeting the IGF system.

Background: Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling., Methods: By comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided., Results: EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways., Conclusion: This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Little is known about the cellular biology of fat surrounding the human heart. In this study, we obtained paired samples of epicardial fat, the visceral fat depot attached to the heart, and subcutaneous skin fat from patients undergoing open heart surgery to test the hypothesis that human epicardial fat cells differentially express bioactive molecules that have the potential to affect cardiac function. First, we characterized the free fatty acids (FFAs), adipocytokines, and growth factors secreted by isolated adipocytes and preadipocytes in cell culture. There was little to distinguish the fat cell secretory products in terms of FFAs and adipocytokines. The most striking finding was that preadipocytes from epicardial adipose tissue expressed high levels of pregnancy-associated plasma protein-A (PAPP-A), a novel metalloproteinase that enhances local insulin-like growth factor (IGF) action through cleavage of inhibitory IGF binding protein-4 (IGFBP-4). PAPP-A levels were 15-fold higher in conditioned medium from epicardial preadipocytes than from subcutaneous preadipocytes (P < 0.0001). PAPP-A was not expressed in mature adipocytes. Next we determined whether PAPP-A could affect IGF-I signaling in a human cardiomyocyte cell line. IGF-I activated receptor-mediated auto-phosphorylation, and this was blocked by wild-type and protease-resistant IGFBP-4. Addition of PAPP-A induced cleavage of wild-type, but not protease-resistant, IGFBP-4 thereby restoring IGF-I action. A proteolytically defective PAPP-A had no effect. IGF-I receptor-mediated signaling through the phosphatidylinositol 3-kinase pathway was similarly inhibited by IGFBP-4 and restored by PAPP-A. Thus, human epicardial fat cells differentially express PAPP-A, which has the potential to affect IGF signaling in the heart., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Although implicated in cardiovascular disease, little is known about the fat surrounding the heart. In humans, epicardial fat is the visceral fat depot of the heart, which directly contacts the myocardium. This strategically placed fat depot is thought to produce bioactive molecules that could affect cardiac function. A major limitation in understanding the biology of epicardial fat is its restricted access in humans and its seeming absence in commonly-used experimental animal models. Although laboratory mice do not have epicardial fat per se, they do have a fat depot around the heart. In this study, we found that mouse pericardial fat has the molecular signature, small adipocyte size, and resistance to differentiation consistent with visceral fat. In addition, we show that mouse pericardial fat is regulated by pregnancy-associated plasma protein-A (PAPP-A), a key modulator of local insulin-like growth factor bioavailability. PAPP-A is highly expressed in mouse pericardial fat at levels equivalent to those in mesenteric visceral fat and 10-fold higher than in subcutaneous inguinal fat (P = .0003). Cultured pre-adipocytes isolated from pericardial fat show 2-fold increased PAPP-A secretion compared to pre-adipocytes isolated from inguinal fat. Furthermore, PAPP-A knock-out mice fed a high fat diet for 20 weeks have significantly reduced pericardial fat (by 60%; P < .0001) compared to wild-type littermates. There was no significant difference in inguinal fat between wild-type and PAPP-A knock-out mice. These data characterize a new mouse model of visceral-like pericardial fat and lay a foundation for understanding its role in human heart disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The zinc metalloproteinase, PAPP-A, enhances local insulin-like growth factor (IGF) action through cleavage of inhibitory IGF-binding proteins, thereby increasing IGF available for IGF receptor-mediated cell proliferation, migration and survival. In many tumors, enhanced IGF receptor signaling is associated with tumor growth, invasion and metastasis. We will first discuss PAPP-A structure and function, and post-translational inhibitors of PAPP-A expression or proteolytic activity. We will then review the evidence supporting an important role for PAPP-A in many cancers, including breast, ovarian and lung cancer, and Ewing sarcoma., (© 2018 Society for Endocrinology.)

Healthy tissue growth depends on a well-controlled and context-appropriate balance of cellular proliferation, cell cycle arrest, and programmed cell death (apoptosis). Disturbance of this balance by activation of oncogenes, inactivation/mutation of tumor suppressor genes, or inhibition of apoptosis can promote tumorigenesis. This mini-review will focus on evidence for the contribution of insulin-like growth factor (IGF) signaling and its regulation by the transcription factor, p53, to tumor development and progression., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pregnancy-associated plasma protein-A (PAPP-A) knockout (KO) mice, generated through homologous recombination in embryonic stem cells, have a significantly increased lifespan compared to wild-type littermates. However, it is unknown whether this longevity advantage would pertain to PAPP-A gene deletion in adult animals. In the present study, we used tamoxifen (Tam)-inducible Cre recombinase-mediated excision of the floxed PAPP-A (fPAPP-A) gene in mice at 5 months of age. fPAPP-A mice, which were either positive (pos) or negative (neg) for Tam-Cre, received Tam treatment with quarterly boosters. Only female mice could be used with this experimental design. fPAPP-A/neg and fPAPP-A/pos mice had similar weights at the start of the experiment and showed equivalent weight gain. We found that fPAPP-A/pos mice had a significant extension of life span (P = 0.005). The median life span was increased by 21% for fPAPP-A/pos compared to fPAPP-A/neg mice. Analysis of mortality in life span quartiles indicated that the proportion of deaths of fPAPP-A/pos mice were lower than fPAPP-A/neg mice at young adult ages (P = 0.002 for 601-800 days) and higher than fPAPP-A/neg mice at older ages (P = 0.004 for >1000 days). Thus, survival curves and age-specific mortality indicate that female mice with knockdown of PAPP-A gene expression as adults have an extended healthy life span., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Pregnancy-associated plasma protein-A (PAPP-A) is a proteolytic enzyme that was discovered to increase local insulin-like growth factor (IGF) availability for receptor activation through cleavage of inhibitory IGF binding proteins (IGFBPs). Reduced IGF signaling has been associated with increased lifespan and healthspan. Therefore, inhibition of PAPP-A represents a novel approach to indirectly decrease the availability of bioactive IGF. Here, we will review data in support of PAPP-A as a therapeutic target to promote healthy longevity., (© 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Advanced atherosclerotic lesions contain senescent cells, but the role of these cells in atherogenesis remains unclear. Using transgenic and pharmacological approaches to eliminate senescent cells in atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr -/- ) mice, we show that these cells are detrimental throughout disease pathogenesis. We find that foamy macrophages with senescence markers accumulate in the subendothelial space at the onset of atherosclerosis, where they drive pathology by increasing expression of key atherogenic and inflammatory cytokines and chemokines. In advanced lesions, senescent cells promote features of plaque instability, including elastic fiber degradation and fibrous cap thinning, by heightening metalloprotease production. Together, these results demonstrate that senescent cells are key drivers of atheroma formation and maturation and suggest that selective clearance of these cells by senolytic agents holds promise for the treatment of atherosclerosis., (Copyright © 2016, American Association for the Advancement of Science.)

Insulin-like growth factors (IGFs) are implicated in the development of diabetic nephropathy (DN) and are shown to increase proliferation and extracellular matrix production in mesangial cells. The IGF system is complex and is composed of ligands, receptors, six binding proteins (IGF BPs) and a novel zinc metalloproteinase - pregnancy-associated plasma protein (PAPP)-A. PAPP-A increases the local bioavailability of IGF through the cleavage of IGF BP-4. Mesangial expansion is a major component of DN, and PAPP-A is shown to be increased in the glomeruli of patients with DN. Therefore, we determined the expression of PAPP-A and components of the IGF system in normal human mesangial cells (HMCs) and their regulation by factors known to be involved in DN. Under basal conditions, HMCs expressed PAPP-A, IGF1 receptor and all six IGF BPs. Interleukin (IL)-1β was the most potent stimulus for PAPP-A expression (5-fold) followed by tumor necrosis factor (TNF)-α (2.5-fold). This PAPP-A was secreted, cell associated and proteolytically active. IL1β also increased IGF BP-1expression (3-fold) with either reduction or no effect on other IGF BPs. Generally, TNF-α treatment decreased IGF BP expression. No treatment effect on PAPP-A or IGF BPs was seen with IL6, IGFs, advanced glycation end products or prolonged hyperglycemia. In addition, stimulation of HMCs with IGF1 alone or IGF1 complexed to wild-type, but not protease-resistant, IGF BP-4 led to increased [(3)H]-thymidine incorporation. In conclusion, these novel findings of PAPP-A and its regulation by proinflammatory cytokines, as well as the comprehensive analysis of the IGF system regulation in HMCs, suggest a mechanism by which inflammatory states such as DN can impact IGF activity in the kidney., (© 2016 Society for Endocrinology.)

Why do studies on insulin-like growth factors (IGFs) and IGF signaling seem so contradictory? The answer is "It depends". This mini- review will explore a few of the factors that are likely to contribute to a seemingly confusing message. Most of the evidence comes from experimental animal models., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multiple myeloma (MM) is a highly heterogeneous plasma cell malignancy. The MM cells reside in the bone marrow (BM), where reciprocal interactions with the BM niche foster MM cell survival, proliferation, and drug resistance. As in most cancers, the insulin-like growth factor (IGF) system has been demonstrated to play a key role in the pathogenesis of MM. The IGF system consists of IGF ligands, IGF receptors, IGF binding proteins (IGFBPs), and IGFBP proteases and contributes not only to the survival, proliferation, and homing of MM cells, but also MM-associated angiogenesis and osteolysis. Furthermore, increased IGF-I receptor (IGF-IR) expression on MM cells correlates with a poor prognosis in MM patients. Despite the prominent role of the IGF system in MM, strategies targeting the IGF-IR using blocking antibodies or small molecule inhibitors have failed to translate into the clinic. However, increasing preclinical evidence indicates that IGF-I is also involved in the development of drug resistance against current standard-of-care agents against MM, including proteasome inhibitors, immunomodulatory agents, and corticoids. IGF-IR targeting has been able to overcome or revert this drug resistance in animal models, enhancing the efficacy of standard-of-care agents. This finding has generated renewed interest in the therapeutic potential of IGF-I targeting in MM. The present review provides an update of the impact of the different IGF system components in MM and discusses the diagnostic and therapeutic potentials., Competing Interests: The authors declare no potential conflict of interest.

Mice deficient in pregnancy-associated plasma protein-A (PAPP-A) have extended lifespan associated with decreased incidence and severity of degenerative diseases of age, such as cardiomyopathy and nephropathy. In this study, the effect of PAPP-A deficiency on aging skeletal muscle was investigated. Whole-genome expression profiling was performed on soleus muscles from 18-month-old wild-type (WT) and PAPP-A knock-out (KO) mice of the same sex and from the same litter ('womb-mates') to identify potential mechanisms of skeletal muscle aging and its retardation in PAPP-A deficiency. Top genes regulated in PAPP-A KO compared to WT muscle were associated with increased muscle function, increased metabolism, in particular lipid metabolism, and decreased stress. Fiber cross-sectional area was significantly increased in solei from PAPP-A KO mice. In vitro contractility experiments indicated increased specific force and decreased fatigue in solei from PAPP-A KO mice. Intrinsic mitochondrial oxidative capacity was significantly increased in skeletal muscle of aged PAPP-A KO compared to WT mice. Moreover, 18-month-old PAPP-A KO mice exhibited significantly enhanced endurance running on a treadmill. Thus, PAPP-A deficiency in mice is associated with indices of healthy skeletal muscle function with age., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Aim: The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) has been suggested as a proatherogenic molecule by its ability to locally increase insulin-like growth factor signaling. Stanniocalcin-2 (STC2) was recently discovered to be a potent inhibitor of PAPP-A activity, but has not previously been implicated in vascular disease. The aim of this study was to substantiate the interaction between PAPP-A and STC2 as a potential local regulatory mechanism in the artery wall., Methods and Results: We found that PAPP-A is secreted from cultured primary smooth muscle cells obtained from human aortas as a covalent complex with STC2, devoid of proteolytic activity. Extracts of human carotid atherosclerotic plaques contain both complexed and uncomplexed PAPP-A, and we show by immunohistochemistry that PAPP-A and STC2 are present in the tissue throughout early human lesion development. We then used adeno-associated virus-mediated expression of STC2 to increase the fraction of PAPP-A present in the inhibited state and found that it decreased the development of atherosclerosis by 47% (P = 0.0005) in apolipoprotein E-deficient mice challenged with a Western type diet compared to controls., Conclusions: This study is the first to suggest the involvement of STC2 in regulating PAPP-A activity during the development of atherosclerosis. Furthermore, we demonstrate that lesion development can be inhibited in an experimental model by driving the balance towards inhibited PAPP-A., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration.

Mutations in multiple genes of the growth hormone/IGF-I axis have been identified in syndromes marked by growth failure. However, no pathogenic human mutations have been reported in the six high-affinity IGF-binding proteins (IGFBPs) or their regulators, such as the metalloproteinase pregnancy-associated plasma protein A2 (PAPP-A2) that is hypothesized to increase IGF-I bioactivity by specific proteolytic cleavage of IGFBP-3 and -5. Multiple members of two unrelated families presented with progressive growth failure, moderate microcephaly, thin long bones, mildly decreased bone density and elevated circulating total IGF-I, IGFBP-3, and -5, acid labile subunit, and IGF-II concentrations. Two different homozygous mutations in PAPPA2, p.D643fs25* and p.Ala1033Val, were associated with this novel syndrome of growth failure. In vitro analysis of IGFBP cleavage demonstrated that both mutations cause a complete absence of PAPP-A2 proteolytic activity. Size-exclusion chromatography showed a significant increase in IGF-I bound in its ternary complex. Free IGF-I concentrations were decreased. These patients provide important insights into the regulation of longitudinal growth in humans, documenting the critical role of PAPP-A2 in releasing IGF-I from its BPs., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

The metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), has been implicated in the development of cardiovascular disease in humans and mouse models. In the latter, genetic deletion or overexpression of PAPP-A confirmed a major role for PAPP-A in atherosclerosis. In this study, we tested the hypothesis that targeting PAPP-A proteolytic activity by an inhibitory monoclonal antibody (mAb-PA) reduces atherosclerotic plaque progression. Apolipoprotein E knock-out mice on high-fat diet were treated with mAb-PA or isotype control. Control mice had a 10-fold increase in aortic plaque after 10 weeks. Aortic plaque burden was reduced by ∼ 70% in mice treated with mAb-PA (P = 0.0002). Treatment was efficacious even in the face of elevated cholesterol and triglycerides. This study demonstrates proof-of-principle and provides feasibility for a novel therapeutic strategy to inhibit atherosclerotic plaque burden by selective targeting of PAPP-A.

Naked mole-rats (Heterocephalus glaber) (NMRs) are the longest living rodents known. They show negligible senescence, and are resistant to cancers and certain damaging effects associated with aging. The insulin-like growth factors (IGFs) have pluripotent actions, influencing growth processes in virtually every system of the body. They are established contributors to the aging process, confirmed by the demonstration that decreased IGF signaling results in life-extending effects in a variety of species. The IGFs are likewise involved in progression of cancers by mediating survival signals in malignant cells. This report presents a full characterization of the IGF system in the NMR: ligands, receptors, IGF binding proteins (IGFBPs), and IGFBP proteases. A particular emphasis was placed on the IGFBP protease, pregnancy-associated plasma protein-A (PAPP-A), shown to be an important lifespan modulator in mice. Comparisons of IGF-related genes in the NMR with human and murine sequences indicated no major differences in essential parts of the IGF system, including PAPP-A. The protease was shown to possess an intact active site despite the report of a contradictory genome sequence. Furthermore, PAPP-A was expressed and translated in NMRs cells and retained IGF-dependent proteolytic activity towards IGFBP-4 and IGF-independent activity towards IGFBP-5. However, experimental data suggest differential regulatory mechanisms for PAPP-A expression in NMRs than those described in humans and mice. This overall description of the IGF system in the NMR represents an initial step towards elucidating the complex molecular mechanisms underlying longevity, and how these animals have evolved to ensure a delayed and healthy aging process.

Intermittent parathyroid hormone (PTH) is a potent anabolic therapy for bone, and several studies have implicated local insulin-like growth factor (IGF) signaling in mediating this effect. The IGF system is complex and includes ligands and receptors, as well as IGF binding proteins (IGFBPs) and IGFBP proteases. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease expressed by osteoblasts in vitro that has been shown to enhance local IGF action through cleavage of inhibitory IGFBP-4. This study was set up to test two specific hypotheses: 1) Intermittent PTH treatment increases the expression of IGF-I, IGFBP-4 and PAPP-A in bone in vivo, thereby increasing local IGF activity. 2) In the absence of PAPP-A, local IGF activity and the anabolic effects of PTH on bone are reduced. Wild-type (WT) and PAPP-A knock-out (KO) mice were treated with 80 μg/kg human PTH 1-34 or vehicle by subcutaneous injection five days per week for six weeks. IGF-I, IGFBP-4 and PAPP-A mRNA expression in bone were significantly increased in response to PTH treatment. PTH treatment of WT mice, but not PAPP-A KO mice, significantly increased expression of an IGF-responsive gene. Bone mineral density (BMD), as measured by DEXA, was significantly decreased in femurs of PAPP-A KO compared to WT mice with PTH treatment. Volumetric BMD, as measured by pQCT, was significantly decreased in femoral midshaft (primarily cortical bone), but not metaphysis (primarily trabecular bone), of PAPP-A KO compared to WT mice with PTH treatment. These data suggest that stimulation of PAPP-A expression by intermittent PTH treatment contributes to PTH bone anabolism in mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The usefulness of circulating pregnancy-associated plasma protein-A (PAPP-A) as a biomarker for acute coronary syndrome (ACS) is widely debated. We used the pig as a model to assess PAPP-A dynamics in the setting of myocardial ischemia. Induction of myocardial ischemia by ligation of the left anterior descending (LAD) coronary artery caused a systemic rise in PAPP-A. However, the ischemic myocardium was excluded as the source of PAPP-A. Interestingly, induction of ischemia in peripheral tissues by ligation of the left femoral artery caused a systemic rise in PAPP-A originating from the left hind limb. This is the first study to demonstrate PAPP-A elevations in the absence of atherosclerosis or heparin during myocardial ischemia. Our findings thus add to the current discussion of the usefulness of PAPP-A as a biomarker for ACS.

Pregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF) action through proteolysis of IGF-binding protein (IGFBP)-4. In experimental animals, PAPP-A accelerates ovarian tumor growth by this mechanism. To investigate the effect of PAPP-A in humans, we compared serum and ascites from 22 women with ovarian carcinoma. We found that ascites contained 46-fold higher PAPP-A levels as compared to serum (P < 0.001). The majority (80%) of PAPP-A was enzymatically active. This is supported by the finding that ascites contained more cleaved than intact IGFBP-4 (P < 0.03). Ascites was more potent than serum in activating the IGF-I receptor (IGF-IR) in vitro (+31%, P < 0.05); in 8 of 22 patients by more than two-fold. In contrast, ascites contained similar levels of immunoreactive IGF-I, and lower levels of IGF-II (P < 0.001). Immunohistochemistry demonstrated the presence of IGF-IR in all but one tumor, whereas all tumors expressed PAPP-A, IGFBP-4, IGF-I and IGF-II. Addition of recombinant PAPP-A to ascites increased the cleavage of IGFBP-4 and enhanced IGF-IR activation (P < 0.05). In conclusion, human ovarian tumors express PAPP-A, IGFBP-4 and IGFs and these proteins are also present in ascites. We suggest that both soluble PAPP-A in ascites and tissue-associated PAPP-A serve to increase IGF bioactivity and, thereby, to stimulate IGF-IR-mediated tumor growth.

Pregnancy-associated plasma protein-A (PAPP-A) serves to increase local insulin-like growth factor (IGF) stimulation of proliferation and differentiation in many tissues through proteolysis of inhibitory IGF-binding proteins. The purpose of this study was to investigate the effects of PAPP-A on tendon structure and mechanical properties. A total of 30 tails from 6-month-old mice were tested with 10 tails in each of following groups: PAPP-A knockout (KO), skeletal-specific PAPP-A overexpressing transgenic (Tg) and wild type (WT). Morphologically, the total tail cross-sectional area (CSA), individual tissue CSAs of bone, muscle and tendon, and fascicle diameter were measured. A fascicle pullout test was performed to assess stiffness and strength of interfascicular structures. Fascicles were mechanically characterized through low and high displacement rate uniaxial tension tests providing modulus at each rate, hysteresis area and stress relaxation ratio. The KO mice had a smaller total tail CSA (p<0.05), fascicle diameter (p<0.05), absolute tendon CSA (p<0.05), fast and slow stiffness (p<0.05 for both) and larger hysteresis area (p<0.05) compared to WT and Tg mice. On the other hand, the Tg mice had a larger fascicle diameter (p<0.05), absolute tendon CSA (p<0.05), higher interfascicular strength and stiffness (p<0.05) and lower fascicular modulus at low displacement rates (p<0.05) compared to WT and KO mice. Tg mice also had larger total tail CSA area (p<0.05) and smaller hysteresis area (p<0.05) than KO mice, and larger normalized tendon CSA (p<0.05) than WT mice. Based on these data, we conclude that PAPP-A affects fascicle structure, thereby affecting tendon phenotype., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Obesity is on the rise in westernized countries, and visceral obesity in particular is associated with enhanced risk of developing metabolic disease and accelerated aging. Various dietary restriction regimens have been shown to extend healthy lifespan in a variety of species. However, identification of alternative approaches that could be more acceptable to humans is actively being pursued. We have shown previously that mice deficient in pregnancy-associated plasma protein-A (PAPP-A) have an extended healthy lifespan on a regular chow diet. In this study, we determined the lifespan of PAPP-A knock-out (KO) and wild-type (WT) littermates fed a high fat diet (HFD) starting at 12 months of age. PAPP-A KO and WT mice had equivalent weight gain as measured over 25 weeks on HFD. However, PAPP-A KO mice on HFD had a significant increase in lifespan (P=0.018). Body composition and tissue pathology were assessed in a separate cohort of mice after 30 weeks on HFD. Percent body fat was equivalent in the two groups. However, there was a decrease in visceral fat depot weights and an increase in serum adiponectin levels in PAPP-A KO compared to WT mice. Major pathological differences were seen in kidney, heart and testes, with PAPP-A KO mice having little, if any, evidence of inflammation, mineralization, or degeneration in these tissues compared to WT mice. Thus, PAPP-A is a novel drug target with the potential to promote healthy longevity without a need for dietary restriction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The majority of ovarian cancer patients acquire resistance to standard platinum chemotherapy and novel therapies to reduce tumor burden and ascites accumulation are needed. Pregnancy-associated plasma protein-A (PAPP-A) plays a key role in promoting insulin-like growth factor (IGF) pathway activity, which directly correlates to ovarian cancer cell transformation, growth, and invasiveness. Herein, we evaluate PAPP-A expression in tumors and ascites of women with ovarian cancer, and determine the antitumor efficacy of a neutralizing monoclonal PAPP-A antibody (mAb-PA) in ovarian cancer using primary patient ovarian tumorgrafts ("Ovatars"). PAPP-A mRNA expression in patient ovarian tumors correlated with poor outcome and was validated as a prognostic surrogate in Ovatar tumors. Following confirmation of mAb-PA bioavailability and target efficacy in vivo, the antitumor efficacy of mAb-PA in multiple Ovatar tumor models was examined and the response was found to depend on PAPP-A expression. Strikingly, the addition of mAb-PA to standard platinum chemotherapy effectively sensitized platinum-resistant Ovatar tumors. PAPP-A protein in ascites was also assessed in a large cohort of patients and very high levels were evident across the entire sample set. Therefore, we evaluated targeted PAPP-A inhibition as a novel approach to managing ovarian ascites, and found that mAb-PA inhibited the development, attenuated the progression, and induced the regression of Ovatar ascites. Together, these data indicate PAPP-A as a potential palliative and adjunct therapeutic target for women with ovarian cancer., (©2015 American Association for Cancer Research.)

Objective: Pregnancy-associated plasma protein-A (PAPP-A) is a zinc metalloproteinase in the insulin-like growth factor system that is expressed by tissues outside of pregnancy and involved in normal and dysregulated growth. PAPP-A has been implicated in several cancers. However, studies of PAPP-A expression in breast cancer are limited. In this study, we assessed PAPP-A expression in different subtypes of human malignant breast cancer. Design Formalin-fixed paraffin-embedded tumor samples from 46 female patients with invasive breast cancer were divided into five defined groups [using markers for HER2, estrogen receptor, progesterone receptor, proliferation] that roughly correlate with molecularly defined subtypes (luminal A, luminal B, luminal/HER2 +, HER2 +, triple negative). These samples were analyzed for PAPP-A expression by immunohistochemistry., Results: PAPP-A staining in tumor tissue was detected in 45 of 46 specimens. There were significantly greater extent and intensity of PAPP-A expression in luminal B specimens with high proliferation index than luminal A specimens (P = 0.01). However, there were no differences between specimens positive or negative for HER2 (P = 0.14) or positive and negative for estrogen receptor (P = 0.31)., Conclusion: PAPP-A was detected in almost all breast cancer specimens and a more intense and greater extent of its expression was associated with luminal B specimens compared to luminal A specimens. The role of PAPP-A in breast cancer prognosis, and possibly therapeutics, warrants further investigation.

Unlabelled: Mice deficient in pregnancy-associated plasma protein-A (PAPP-A), an IGF binding protein protease, have been shown to be resistant to experimentally induced atherosclerosis and diabetic nephropathy, and, in the laboratory environment, live 30-40% longer than wild-type littermates in association with delayed incidence and occurrence of age-related neoplasms and degenerative diseases., Objective: PAPP-A is highly expressed in the cerebellum and hippocampus of the mouse brain. Therefore, the studies presented here were aimed at determining motor behavior, learning and retention in PAPP-A knock-out (KO) mice compared to wild-type (WT) littermates with age., Design: Balance and coordination were assessed using an accelerating rotarod; learning and memory were assessed in a Stone T-maze., Results: Time on the rotarod decreased with age but there was no significant difference between PAPP-A KO and WT mice at any of the testing ages. Latency to reach the goal box and number of errors committed in the Stone T-maze did not change with age and there were no significant differences between PAPP-A KO and WT mice., Conclusion: Lack of PAPP-A in mice did not impact central regulation of coordination, learning or memory., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Pregnancy-associated plasma protein-A (PAPP-A) is a novel zinc metalloproteinase that functions in many systems outside of pregnancy. Data in both humans and mice suggest a role for PAPP-A in aging and age-related diseases. However, our knowledge of tissue-specific PAPP-A expression and possible changes in this expression with age is limited. Thus, the aim of this study was to determine PAPP-A mRNA expression in multiple tissues with age in both male and female mice using real-time PCR. These included the heart, liver, kidney, bone, fat, skeletal muscle, gonads, brain, thymus and spleen. In young mice, PAPP-A mRNA was expressed at relatively high levels in all tissues examined except for liver. The only difference in expression between males and females was seen in the kidney, subcutaneous fat and gonads. The highest PAPP-A mRNA expression levels were found in visceral fat and these were 10-fold higher than in subcutaneous fat. PAPP-A expression significantly increased with age in kidney, brain and gonads. PAPP-A expression significantly deceased with age in bone and skeletal muscle. In the thymus, PAPP-A mRNA showed a biphasic response with age. There were no age-related changes in PAPP-A expression seen in any of the other tissues examined. Expression of IGFBP-5 mRNA, a marker of insulin-like growth factor-I (IGF-I) bioactivity known to be regulated by PAPP-A, paralleled the changes in PAPP-A expression with age in kidney, bone, skeletal muscle and thymus. Thus, tissue-specific PAPP-A expression in mice is differentially affected during aging, and may regulate local IGF-I bioactivity in certain tissues., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fat distribution differs between individuals, and those with visceral fat predominance develop metabolic profiles that increase the risk of adverse cardiovascular events. This is due, in part, to the proinflammatory state associated with visceral obesity as well as depot-specific adipogenesis. The IGF system is important in adipose tissue development and metabolic function. Pregnancy-associated plasma protein A (PAPPA) is a novel zinc metalloproteinase that regulates local IGF availability. The first aim of this study was to characterize PAPPA mRNA and protein expression in primary cultures of human preadipocytes isolated from omental, mesenteric, and subcutaneous depots. PAPPA expression was significantly increased in omental preadipocytes compared with mesenteric and subcutaneous preadipocytes. The second aim of this study was to investigate the factors regulating PAPPA expression, focusing on proinflammatory cytokines and resveratrol that have been shown to have negative and positive effects, respectively, on metabolism and diet-induced obesity. Treatment of cultured primary human preadipocytes with tumor necrosis factor α and interleukin 1β led to significant increases in PAPPA expression. Activated pathways mediating cytokine-induced PAPPA expression include the nuclear factor κB pathway and the MAPK family, particularly c-Jun NH2-terminal kinase and p38 MAPK. Resveratrol, a polyphenolic compound with beneficial cardiometabolic effects, significantly downregulated PAPPA expression under basal and stimulated conditions. Effects of resveratrol on PAPPA appeared to be mediated through pathways independent of silent mating type information regulation 2 homolog 1 (SIRT1) and AMP kinase activation. Depot-specific PAPPA expression in human preadipocytes may contribute to a depot-specific function., (© 2014 Society for Endocrinology.)

IGF1 and IGF2 are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF-binding proteins (IGFBP1-IGFBP6) are primary regulators of IGF half-life and receptor availability. Generally, the binding of IGFBPs inhibits IGF receptor activation. However, it has been shown that IGFBP2 in complex with IGF2 (IGF2/IGFBP2) stimulates osteoblast function in vitro and increases skeletal mass in vivo. IGF2 binding to IGFBP2 greatly increases the affinity for 2- or 3-carbon O-sulfated glycosaminoglycans (GAGs), e.g. heparin and heparan sulfate, which is hypothesized to preferentially and specifically target the IGF2/IGFBP2 complex to the bone matrix. In order to obtain a more detailed understanding of the interactions between the IGF2/IGFBP2 complex and GAGs, we investigated heparin-binding properties of IGFBP2 and the IGF2/IGFBP2 complex in a quantitative manner. For this study, we mutated key positively charged residues within the two heparin-binding domains (HBDs) in IGFBP2 and in one potential HBD in IGF2. Using heparin affinity chromatography, we demonstrate that the two IGFBP2 HBDs contribute differentially to GAG binding in free IGFBP2 and the IGF2/IGFBP2 protein complex. Moreover, we identify a significant contribution from the HBD in IGF2 to the increased IGF2/IGFBP2 heparin affinity. Using molecular modeling, we present a novel model for the IGF2/IGFBP2 interaction with heparin where all three proposed HBDs constitute a positively charged and surface-exposed area that would serve to promote the increased heparin affinity of the complex compared with free intact IGFBP2., (© 2014 Society for Endocrinology.)

Pregnancy-associated plasma protein-A (PAPP-A) is a novel zinc metalloproteinase implicated in cardiovascular disease. The aim of this study was to determine whether a reduction in PAPP-A expression in the adult affects the progression of established atherosclerotic plaque. Apolipoprotein E-null mice were fed a high-fat diet for 5 weeks to initiate early-stage plaque development before tamoxifen-inducible, Cre recombinase-mediated excision of the floxed PAPP-A gene. High-fat feeding was continued, and after 10 weeks the aorta and brachiocephalic artery were harvested for atherosclerotic plaque analyses of overall burden and morphology, respectively. An inducible decrease in PAPP-A gene expression significantly inhibited atherosclerotic plaque progression as assessed by a 70% reduction in plaque burden in the aorta (P = .012) without an effect on the elevated circulating levels of cholesterol and triglycerides in this model. Furthermore, this reduction in PAPP-A prevented the development of advanced plaque with necrotic cores and buried fibrous caps in the brachiocephalic artery. These data indicate PAPP-A as a potential target to limit progression of established atherosclerotic plaque.

The insulin-like growth factor (IGF) signaling pathway is involved in certain human cancers, and the feasibility of directly targeting the IGF receptor has been actively investigated. However, recent evidence from clinical trials suggests that this approach can be problematic. We have developed an alternative strategy to indirectly inhibit the IGF signaling by targeting the metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). PAPP-A associated with the cell surface cleaves IGF binding protein-4 (IGFBP-4), when IGF is bound to IGFBP-4, and thereby increases IGF bioavailability for receptor activation in an autocrine/paracrine manner. We hypothesized that inhibition of PAPP-A would suppress excessive local IGF signaling in tissues where this is caused by increased PAPP-A proteolytic activity. To test this hypothesis, we developed an inhibitory monoclonal antibody, mAb 1/41, which targets a unique substrate-binding exosite of PAPP-A. This inhibitor selectively and specifically inhibits proteolytic cleavage of IGFBP-4 with an inhibitory constant (Ki) of 135 pM. In addition, it inhibited intracellular signaling of the IGF receptor (AKT phosphorylation) in monolayers of A549 cells, an IGF-responsive lung cancer-derived cell line found to express high levels of PAPP-A. We further showed that mAb 1/41 is effective towards PAPP-A bound to cell surfaces, and that it is capable of inhibiting PAPP-A activity in vivo. Using a murine xenograft model of A549 cells, we demonstrated that mAb 1/41 administered intraperitoneally significantly inhibited tumor growth. Analysis of xenograft tumor tissue recovered from treated mice showed penetration of mAb 1/41, reduced IGFBP-4 proteolysis, and reduced AKT phosphorylation. Our study provides proof of concept that IGF signaling can be selectively reduced by targeting a regulatory proteinase that functions extracellularly, upstream of the IGF receptor. PAPP-A targeting thus represents an alternative therapeutic strategy for inhibiting IGF receptor signaling.

Accumulation of visceral fat, more so than subcutaneous fat, is strongly associated with severe metabolic complications. However, the factors regulating depot-specific adipogenesis are poorly understood. In this study, we show differential expression of pregnancy-associated plasma protein-A (PAPP-A), a secreted regulator of local insulin-like growth factor (IGF) action, in adipose tissue of mice. PAPP-A mRNA expression was fivefold higher in visceral (mesenteric) fat compared with subcutaneous (inguinal, subscapular), perirenal, and brown fat of mice. To investigate the possible role of depot-specific PAPP-A expression in fat accumulation, wild-type (WT) and PAPP-A knockout (KO) mice were fed a high-fat diet (HFD) for up to 20 wk. Adipocyte size increased in subcutaneous and perirenal depots similarly in WT and PAPP-A KO mice. However, fat cell size and in vivo lipid uptake were significantly reduced in mesenteric fat of PAPP-A KO compared with WT mice. After 20 wk on HFD, phosphorylation of AKT, a downstream signaling intermediate of IGF-I and insulin receptor activation, was significantly decreased by 50% in mesenteric compared with subcutaneous fat in WT mice, but was significantly increased threefold in mesenteric compared with subcutaneous fat in PAPP-A KO mice. This appeared to be because of enhanced insulin-stimulated signaling in mesenteric fat of PAPP-A KO mice. These data establish fat depot-specific expression of PAPP-A and indicate preferential impact of PAPP-A deficiency on visceral fat in the mouse that is associated with enhanced insulin receptor signaling. Thus, PAPP-A may be a potential target for treatment and/or prevention strategies for visceral obesity and related morbidities.

We investigated pregnancy-associated plasma protein-A (PAPP-A) in diabetic nephropathy. Normal human kidney showed specific staining for PAPP-A in glomeruli, and this staining was markedly increased in diabetic kidney. To assess the possible contribution of PAPP-A in the development of diabetic nephropathy, we induced diabetes with streptozotocin in 14-month-old WT and Papp-A knockout (KO) mice. Renal histopathology was evaluated after 4 months of stable hyperglycemia. Kidneys from diabetic WT mice showed multiple abnormalities including thickening of Bowman's capsule (100% of mice), increased glomerular size (80% of mice), tubule dilation (80% of mice), and mononuclear cell infiltration (90% of mice). Kidneys of age-matched non-diabetic WT mice had similar evidence of tubule dilation and mononuclear cell infiltration to those of diabetic WT mice, indicating that these changes were predominantly age-related. However, thickened Bowman's capsule and increased glomerular size appeared specific for the experimental diabetes. Kidneys from diabetic Papp-A KO mice had significantly reduced or no evidence of changes in Bowman's capsule thickening and glomerular size. There was also a shift to larger mesangial area and increased macrophage staining in diabetic WT mice compared with Papp-A KO mice. In summary, elevated PAPP-A expression in glomeruli is associated with diabetic nephropathy in humans and absence of PAPP-A is associated with resistance to the development of indicators of diabetic nephropathy in mice. These data suggest PAPP-A as a potential therapeutic target for diabetic nephropathy.

Pregnancy-associated plasma protein-A (PAPP-A) enhances local IGF signaling through its ability to proteolyze inhibitory IGF binding proteins. In vivo, PAPP-A (like IGF) appears to exhibit antagonistic pleiotropy; ie, it has beneficial effects early in life but detrimental effects later in life. Accordingly, PAPP-A knockout (KO) mice are born as proportional dwarfs and have diminished reproductive vigor and reduced peak bone mass acquisition at puberty. On the other hand, PAPP-A KO mice live approximately 30% longer than their wild-type littermates, with decreased incidence and severity of age-related diseases and resistance to adverse responses of vascular injury. To be able to distinguish the impact of PAPP-A deficiency in the adult from that in early life, we developed a mouse model suitable for inducible Cre recombinase-mediated excision of the PAPP-A gene. In this study, we characterize the conditional PAPP-A KO mouse model for efficacy of tamoxifen-induced floxed PAPP-A excision in various tissues of adult mice and demonstrate a significant (P = .0001) reduction of neointimal formation in these mice after unilateral carotid artery ligation.

As suggested by its name, pregnancy-associated plasma protein-A (PAPP-A) plays an important role in pregnancy and fetal development (Brizot et al., 1996; Lin et al., 1974; Smith et al., 2002). On the opposite end of life's spectrum, recent studies using genetically-engineered mice indicate a newly recognized role for PAPP-A in aging and in the development of age-related disease. These latter studies will be reviewed in this article., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.

Pregnancy-associated plasma protein-A (PAPP-A) functions to increase local IGF-I bioactivity. In this study, we used transgenic mice that constitutively express human PAPP-A in arterial smooth muscle to test the hypothesis that overexpression of PAPP-A enhances vascular smooth muscle cell (SMC) response to IGF-I in vivo. PAPP-A transgenic (Tg) and wild-type (WT) mice underwent unilateral carotid ligation, a model of injury-induced SMC hyperplasia and neointimal formation. In both WT and PAPP-A Tg mice, endogenous PAPP-A mRNA expression showed peak elevation 5 days after carotid ligation. However, PAPP-A Tg mice had 70-75% less neointima than WT at 5 and 10 days postligation, with a significant reduction in occlusion of the ligated artery. WT and PAPP-A Tg mice had equivalent increases in medial area and vessel remodeling postligation. There was little change in medial area and no evidence of neointima in the contralateral carotid of WT or PAPP-A Tg mice. Both WT and PAPP-A Tg carotids exhibited signs of dedifferentiation of SMC, which precedes the increase in proliferation and migration that results in neointimal formation. However, the number of proliferating cells in the media and neointima of the ligated PAPP-A Tg artery was reduced by 90% on day 5 postsurgery compared with WT. This decrease was associated with a significant decrease in an in vivo marker of IGF-I bioactivity and reduced IGF-I-stimulated receptor phosphorylation ex vivo. These data suggest differential effects of chronic (transgenic) and transient (endogenous) PAPP-A expression on neointimal formation following vascular injury that may be due in part to the differential impact on IGF-I signaling.