The brown bear as a translational model for sedentary lifestyle-related diseases.

Sedentary lifestyle accelerates biological ageing, is a major risk factor for developing metabolic syndrome and is associated with cardiovascular disease, diabetes mellitus, kidney failure, sarcopenia and osteoporosis. In contrast to the linear path to worsening health in humans with metabolic syndrome, brown bears have developed a circular metabolic plasticity enabling these animals to tolerate obesity and a 'sedentary lifestyle' during hibernation and exit the den metabolically healthy in spring. Bears are close to humans physiology wise, much closer than rodents, the preferred experimental animals in medical research, and may better serve as translational model to develop treatments for lifestyle-related diseases. In this review, aspects of brown bear hibernation survival strategies are outlined and conceivable experimental strategies to learn from bears are described.

Antimicrobial peptide CAP18 and its effect on Yersinia ruckeri infections in rainbow trout Oncorhynchus mykiss (Walbaum): comparing administration by injection and oral routes.

The antimicrobial peptide CAP18 has been demonstrated to have a strong in vitro bactericidal effect on Yersinia ruckeri, but its activity in vivo has not been described. In this work, we investigated whether CAP18 protects rainbow trout Oncorhynchus mykiss (Walbaum) against enteric red mouth disease caused by this pathogen either following i.p. injection or by oral administration (in feed). It was found that injection of CAP18 into juvenile rainbow trout before exposure to Y. ruckeri was associated with lowered mortality compared to non-medicated fish although it was less effective than the conventional antibiotic oxolinic acid. Oral administration of CAP18 to trout did not prevent infection. The proteolytic effect of secretions on the peptide CAP18 in the fish gastrointestinal tract is suggested to account for the inferior effect of oral administration.

A robust immunoassay for pregnancy-associated plasma protein-A2 based on analysis of circulating antigen: establishment of normal ranges in pregnancy.

Pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, two homologous metzincin metalloproteases, are both tightly linked to regulation within the insulin-like growth factor (IGF) system because of their specific cleavage of IGF binding proteins. Recent studies suggest that PAPP-A may be involved in clinical conditions related to unwanted cellular growth, and the circulating levels of PAPP-A is an established biomarker in prenatal screening for chromosomal abnormalities. Microarray data indicate that PAPP-A2 has potential as a biomarker for pre-eclampsia. However, well-characterized immunological methods of quantification are not available. We therefore developed monoclonal antibodies against recombinant PAPP-A2. The antibodies were epitope mapped against recombinantly expressed chimeras between PAPP-A2 and PAPP-A. Furthermore, circulating PAPP-A2 was immunoaffinity purified and characterized by sequence analysis and mass spectrometry. Unlike PAPP-A, PAPP-A2 is a noncovalent dimer in which each subunit of 1558 amino acids originates from all of the 22 predicted coding exons. A previously hypothesized variant (PAPP-E) does not exist, but low amounts of a C-terminally truncated PAPP-A2 variant was detected. A sensitive and robust ELISA for full-length PAPP-A2 was developed and used to establish normal ranges of PAPP-A2 through pregnancy. The functional sensitivity of this ELISA at 20% CV was 0.08 ng/ml, and the serum concentration of PAPP-A2 was found to increase during pregnancy in agreement with placental synthesis. The existence of this assay will enable an assessment of the biomarker potential of PAPP-A2 in pre-eclampsia as well as other clinical conditions.

Expression of membrane progesterone receptors on human T lymphocytes and Jurkat cells and activation of G-proteins by progesterone.

Although there is significant evidence for progesterone's role as an immunomodulator, nuclear progesterone receptors have not been consistently identified in immune cells. Recently, three new putative membrane progesterone receptors (mPRs), mPRalpha, mPRbeta, and mPRgamma have been described. The objective of this study was to examine whether mPRs are expressed in peripheral blood leukocytes (PBLs) in women of reproductive age, and to further characterize them in T lymphocytes and immortalized T cells (Jurkat cells). Transcripts for mPRalpha and mPRbeta but not mPRgamma, were detected by RT-PCR in PBLs, T lymphocytes, and Jurkat cells. Western blot analysis showed the presence of the mPRalpha and mPRbeta proteins on cell membranes of T lymphocytes and Jurkat cells. Expression of the mPRalpha mRNA was upregulated in the luteal phase of the menstrual cycle in cluster of differentiation (CD)8+, but not in CD4+, T lymphocytes. Radioreceptor assays revealed specific [(3)H]progesterone binding to T- and Jurkat cell membranes (K(d) 4.25 nM) characteristic of steroid membrane receptors. Progesterone activated an inhibitory G-protein (G(i)), suggesting that mPRs are coupled to G(i) in Jurkat cells. These results suggest a potential novel mechanism for progesterone's immunoregulatory function through activation of mPRs.

Slow spontaneous [Ca2+] i oscillations reflect nucleotide release from renal epithelia.

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca(2+)](i) increases in renal epithelia. Spontaneous [Ca(2+)](i) increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 +/- 6.7% (n = 23) of the cells showed spontaneous [Ca(2+)](i) increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca(2+)](i) oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca(2+)](i) oscillatory activity. The association between spontaneous [Ca(2+)](i) increases and nucleotide signalling was further tested in 132-1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca(2+)](i) increases. Transfection with either hP2Y(6) or hP2Y(2) receptors revealed a striking degree of oscillations. Similar spontaneous [Ca(2+)](i) increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y(2) knock out mice (0.050 +/- 0.020 events per second, n = 8) compared to the wild type (0.147 +/- 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca(2+)](i) oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.

Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.

Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE-->ESE, ESE-->MSE, and MSE-->LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy specimens) from subjects who are as normal as possible in a human study and including those with unknown histology, can be classified by their molecular signatures and correspond to known phases of the menstrual cycle with identical results using two independent analytical methods. Also, the results enable global identification of biological processes and molecular mechanisms that occur dynamically in the endometrium in the changing steroid hormone milieu across the menstrual cycle in normo-ovulatory women. The results underscore the potential of gene expression profiling for developing molecular diagnostics of endometrial normalcy and abnormalities and identifying molecular targets for therapeutic purposes in endometrial disorders.

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles.

In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work, we showed that follicular fluid from late dominant equine follicles (35 mm diameter) contains a proteolytic activity against IGFBP-2. First follicular fluid from dominant follicles contained lower levels of native IGFBP-2 than the corresponding serum, as assessed by Western ligand blotting. In contrast, immunoblotting experiments showed much higher levels of a 12 kDa proteolytic fragment in dominant follicular fluid than in the serum. Moreover, equine dominant follicular fluid was able to induce proteolysis of exogenous recombinant bovine (rb)IGFBP-2, this degradation being dose-dependently enhanced by IGFs. The proteolytic activity against IGFBP-2 in equine follicles was partially immunoneutralized by a polyclonal antibody raised against human PAPP-A. Moreover, cleavage of rbIGFBP-2 by equine follicular fluid was dose-dependently inhibited by a peptide derived from the heparin-binding domain of IGFBP-5, as well as by peptides derived from other heparin-binding domain-containing proteins such as connective tissue growth factor, vitronectin and heparin-interacting protein, previously shown to inhibit PAPP-A. Finally, the proteolytic activity was very low in subordinate follicles, was high in both early (25 mm diameter) and late (35 mm diameter) dominant follicles, and was slightly lower in preovulatory follicles recovered 35 h after human chorionic gonadotropin (hCG) treatment.Overall, these data show that in the equine ovary, the selection of dominant follicles is associated with an increase of the proteolytic degradation of IGFBP-2 by PAPP-A, as for IGFBP-4, and potentially other protease(s), probably contributing to the increase in IGF bioavailability. In atretic subordinate follicles, the decrease in the proteolytic degradation of IGFBP-2, probably due in part to a direct inhibition by peptides containing heparin-binding domains, contributes to the increase in IGFBP-2 levels and the decrease in IGF bioavailability. The expression of PAPP-A and IGFBP-2 mRNA during folliculogenesis remain to be investigated in the mare.

Screening for Down's syndrome in early and late first and second trimester using six maternal serum markers.

The efficiency of six maternal serum markers for Down's syndrome (DS), alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), free beta-hCG, pregnancy-associated plasma protein-A (PAPP-A), the proform of eosinophil major basic protein (ProMBP), pregnancy-specific-beta-1-glycoprotein (SP(1)), and combinations thereof, was examined. Discriminant analysis in 156 DS pregnancies and 546 controls defined three effective combinations of serum marker logMoMs (multiples of the median in control samples) in three gestational age windows, i.e. Index I (weeks 7-9) = 0.52 logMoM ProMBP + 0.28 logMoM PAPP-A - logMoM SP(1); Index II (weeks 10-12) = 1.94 logMoM free beta-hCG - logMoM SP(1), and Index III (weeks 15-19) = 0.78 logMoM free beta-hCG + 1.12 logMoM ProMBP - logMoM AFP. The estimated detection rates of indices and age for a false-positive rate (FPR) of 5% were 73% for Index I, 69% for Index II, and 60% for Index III. Including the ultrasound marker nuchal translucency, using a DS at term risk of 1 : 400 as cut-off, the detection rates of the indices increased to 86, 83, and 82% for FPRs of 4.3, 4.1, and 5.8%, respectively. The indices are promising markers for screening for DS.

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation.

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.

Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development.

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Pregnancy-associated plasma protein A as a marker of acute coronary syndromes.

BACKGROUND: Circulating markers indicating the instability of atherosclerotic plaques could have diagnostic value in unstable angina or acute myocardial infarction. We evaluated pregnancy-associated plasma protein A (PAPP-A), a potentially proatherosclerotic metalloproteinase, as a marker of acute coronary syndromes. METHODS: We examined the level of expression of PAPP-A in eight culprit unstable coronary plaques and four stable plaques from eight patients who had died suddenly of cardiac causes. We also measured circulating levels of PAPP-A, C-reactive protein, and insulin-like growth factor I (IGF-I) in 17 patients with acute myocardial infarction, 20 with unstable angina, 19 with stable angina, and 13 controls without atherosclerosis. RESULTS: PAPP-A was abundantly expressed in plaque cells and extracellular matrix of ruptured and eroded unstable plaques, but not in stable plaques. Circulating PAPP-A levels were significantly higher in patients with unstable angina or acute myocardial infarction than in patients with stable angina and controls (P<0.001). A PAPP-A threshold value of 10 mlU per liter identified patients who had acute coronary syndromes with a sensitivity of 89.2 percent and a specificity of 81.3 percent. PAPP-A levels correlated with levels of C-reactive protein and free IGF-I, but not with markers of myocardial injury (troponin I and the MB isoform of creatine kinase). CONCLUSIONS: PAPP-A is present in unstable plaques, and circulating levels are elevated in acute coronary syndromes; these increased levels may reflect the instability of atherosclerotic plaques. PAPP-A is a new candidate marker of unstable angina and acute myocardial infarction.

Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin.

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

Pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor binding protein (IGFBP)-5 independent of IGF: implications for the mechanism of IGFBP-4 proteolysis by PAPP-A.

Pregnancy-associated plasma protein-A (PAPP-A) has recently been identified as the proteinase responsible for cleavage of insulin-like growth factor binding protein (IGFBP)-4, an inhibitor of IGF action, in several biological fluids. Cleavage of IGFBP-4 by PAPP-A is believed to occur only in the presence of IGF. We here report that in addition to IGFBP-4, PAPP-A also cleaves IGFBP-5. Cleavage occurs at one site, between Ser-143 and Lys-144 of IGFBP-5. In the presence of IGF, IGFBP-4 and -5 are cleaved with similar rates by PAPP-A. Interestingly, cleavage of IGFBP-5 by PAPP-A does not require the presence of IGF, but is slightly inhibited by IGF. These findings have implications for the mechanism of proteolysis of IGFBP-4 by PAPP-A, suggesting that IGFBP-4 binds IGF, which then becomes a PAPP-A substrate. Using highly purified, recombinant proteins, we establish that (1) PAPP-A cleavage of IGFBP-4 can occur in the absence of IGF, although the rate of hydrolysis is very slow, and (2) IGF is unable to bind to PAPP-A. We thus conclude that IGF enhances proteolysis by binding to IGFBP-4, not by interaction with PAPP-A, which could not previously be ruled out.

Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum.

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from /=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.

Pregnancy-associated plasma protein-A2 (PAPP-A2), a novel insulin-like growth factor-binding protein-5 proteinase.

A novel metalloproteinase with similarity to pregnancy-associated plasma protein-A (PAPP-A), which we denoted PAPP-A2, has been identified. Through expression in mammalian cells we showed that recombinant PAPP-A2 polypeptide of 1558 residues resulted from processing of a 1791-residue prepro-protein. Unlike PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the members of this family. We showed that PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.

Insulin-like growth factor binding protein-4 protease produced by smooth muscle cells increases in the coronary artery after angioplasty.

Insulin-like growth factor (IGF)-I stimulates vascular smooth muscle cell (VSMC) migration and proliferation, which are fundamental to neointimal hyperplasia in postangioplasty restenosis. IGF-I action is modulated by several high-affinity IGF binding proteins (IGFBPs). IGFBP-4 is the predominant IGFBP produced by VSMCs and is a potent inhibitor of IGF-I action. However, specific IGFBP-4 proteases can cleave IGFBP-4 and liberate active IGF-I. In this study, we document IGFBP-4 protease produced by human and porcine coronary artery VSMCs in culture as pregnancy-associated plasma protein-A (PAPP-A). This was shown by a distinctive IGFBP-4 cleavage pattern, specific inhibition of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition of PAPP-A by monoclonal antibodies. Furthermore, we found a 2-fold increase in IGFBP-4 protease activity in injured porcine VSMC cultures in vitro (P<0.05). We also evaluated IGFBP-4 protease/PAPP-A expression in vivo after coronary artery balloon injury. Twenty-five immature female pigs underwent coronary overstretch balloon injury, and vessels were examined at defined time points after the procedure. Abundant PAPP-A expression was observed in the cytoplasm of medial and neointimal cells 7, 14, and 28 days after angioplasty (P<0.01 vs control). The highest PAPP-A labeling indices were located in the neointima (36.1+/-2.1%) and the media (31.7+/-1.2%) 28 days after injury. Western blot analysis confirmed increased PAPP-A in injured vessels. PAPP-A, a regulator of IGF-I bioavailability through proteolysis of IGFBP-4, is thus expressed by VSMCs in vitro and in restenotic lesions in vivo. These results suggest a possible role for PAPP-A in neointimal hyperplasia.

Quantification and characterization of pregnancy-associated complexes of angiotensinogen and the proform of eosinophil major basic protein in serum and amniotic fluid.

BACKGROUND: The proform of eosinophil major basic protein (ProMBP) exists in serum from pregnant women complexed with a variable fraction of angiotensinogen (Ang). A subfraction further binds complement C3dg in a 2:2:2 complex. The function, physiology, and clinical importance of ProMBP complexes are unknown, and the specific quantification of these complexes has not been possible. METHODS: We developed an ELISA for the ProMBP/Ang complexes, using a monoclonal antibody against ProMBP for capture and a chicken anti-human Ang antiserum for detection. Calibrators were standardized with WHO IRP 78/610 for pregnancy proteins in the assay range 0.95-15.6 mIU/L. RESULTS: The concentrations of ProMBP/Ang complexes in serum of nonpregnant blood donors (n = 79) were log-normally distributed with a central 95th interval of 985-3655 mIU/L. In pregnancy, mean serum concentrations were increased from week 7, and the concentrations reached term concentrations in week 18. ProMBP/Ang complexes eluted in gel filtration as a broad peak with a molecular mass of approximately 230 kDa. The concentration of ProMBP/Ang/C3dg increased during blood coagulation, suggesting that the ProMBP/Ang/C3dg complex may be a marker of complement activation. CONCLUSIONS: ProMBP/Ang complexes are present in serum from nonpregnant persons as well as pregnant women, and the direct assays described here will make it possible to study the biochemistry and the clinical significance of different ProMBP complexes in pathological conditions and pregnancy.

Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor.

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

Evidence that the insulin-like growth factor binding protein-4 protease in human ovarian follicular fluid is pregnancy associated plasma protein-A.

Ovarian insulin-like growth factor binding protein-4 (IGFBP-4) proteolysis is involved in the regulation of follicular development, but until now the identity of the responsible enzyme was unknown. In this study, we identify the IGFBP4 protease in human follicular fluid as pregnancy associated plasma protein-A (PAPP-A) based on distinctive IGFBP-4 cleavage pattern, the same protease inhibitor profile, specific inhibition and immunodepletion of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition by PAPP-A monoclonal antibodies in ELISA. Furthermore, PAPP-A levels in estrogen-dominant and androgen-dominant follicular fluids reflect their IGFBP-4 proteolytic activity. PAPP-A was also secreted by human granulosa cells, the reputed source of IGFBP-4 protease activity in follicular fluid. We have the molecular and biochemical tools to begin to delineate the regulation and biological function of PAPP-A in normal and dysregulated follicular development and atresia.

The proform of eosinophil major basic protein: a new maternal serum marker for Down syndrome.

The proform of eosinophil major basic protein (proMBP), the most abundant protein in the eosinophil specific granule, is synthesized by the placenta and secreted into the maternal circulation, where it is found complex-bound to pregnancy-associated plasma protein-A (PAPP-A) and other proteins. We examined the potential of proMBP as a maternal serum marker for fetal Down syndrome (DS) by determining its maternal serum concentration (MSpMBP) in 25 Down syndrome (DS) pregnancies and 152 control pregnancies in the first trimester, and in 105 DS pregnancies and 156 control pregnancies in the second trimester. The median (95 per cent confidence interval) MSpMBP MoM in DS pregnancies (n=15) was 0.66 (0.49-0.79) in gestational weeks 5-9; 1.06 (0.71-1.97) in weeks 10-12 (n=10) and 1.62 (1.18-1.98) in weeks 14-20 (n=105). Using parameterized receiver operator characteristics analysis for proMBP as a single marker for DS, detection rates (DRs) of 22 per cent and 38 per cent, for false-positive rates (FPRs) of 5 per cent, were found in weeks 5-9 (using MSpMBP/=cut-off), respectively. When age and MSpMBP were used as markers in combination, a DR of 36.8 per cent for an FPR of 5.5 per cent was obtained in weeks 5-9 using a risk cut-off of 1:250. In weeks 14-20 the DR was 48.4 per cent for an FPR of 5.3 per cent using the same risk cut-off. This makes proMBP a marker comparable in diagnostic efficiency to human chorionic gonadotrophin (hCG), and exceeding that of alpha-fetoprotein (AFP) and unconjugated oestriol (uE3), in the second trimester.