Conover, Cheryl A.

Abstract —Insulin-like growth factors I and II (IGF-I and -II) and their regulatory proteins are secreted by cells of the cardiovascular system. They are growth promoters for arterial cells and mediators of cardiovascular disease. IGFs are bound to IGF binding proteins (IGFBPs), which modulate IGF ligand-receptor interaction and consequently to IGF action. IGFBPs are in turn posttranslationally modulated by specific proteases. This dynamic balance (IGFs, IGFBPs, and IGFBP proteases) constitutes the IGF axis and ultimately determines the extent of IGF-dependent cellular effects. Dysregulated actions of this axis influence coronary atherosclerosis through effects on vascular smooth muscle cell growth, migration, and extracellular matrix synthesis in the atherosclerotic plaque. IGF-I promotes macrophage chemotaxis, excess LDL cholesterol uptake, and release of proinflammatory cytokines. Endothelial cells also receive the effects of IGFs stimulating their migration and organization forming capillary networks. Neointimal hyperplasia of restenosis after coronary artery injury is also modulated by the IGF axis. IGFs stimulate vascular smooth muscle cell proliferation and migration to form the neointima and upregulate tropoelastin synthesis after disruption of the elastic layer. Understanding IGF axis regulation establishes a scientific basis for strategies directed to limit or reverse plaque growth and vulnerability in atherosclerosis and in the neointimal hyperplasia of restenosis.

Abstract —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.

Pregnancy-associated plasma protein-A2 (PAPP-A2) is a novel homolog of PAPP-A in the metzincin superfamily. However, compared with the accumulating data on PAPP-A, very little is known about PAPP-A2. In this study, we determined the tissue expression pattern of PAPP-A2 mRNA in wild-type (WT) mice and characterized the phenotype of mice with global PAPP-A2 deficiency. Tissues expressing PAPP-A2 in WT mice were more limited than those expressing PAPP-A. The highest PAPP-A2 mRNA expression was found in the placenta, with abundant expression in fetal, skeletal, and reproductive tissues. Heterozygous breeding produced the expected Mendelian distribution for the pappa2 gene and viable homozygous PAPP-A2 knockout (KO) mice that were normal size at birth. The most striking phenotype of the PAPP-A2 KO mouse was postnatal growth retardation. Male and female PAPP-A2 KO mice had 10 and 25–30% lower body weight, respectively, than WT littermates. Adult femur and body length were also reduced in PAPP-A2 KO mice, but without significant effects on bone mineral density. PAPP-A2 KO mice were fertile, but with compromised fecundity. PAPP-A expression was not altered to compensate for the loss of PAPP-A2 expression, and proteolysis of PAPP-A2's primary substrate, IGF-binding protein-5, was not altered in fibroblasts from PAPP-A2 KO embryos. In conclusion, tissue expression patterns and biological consequences of gene KO indicate distinct physiological roles for PAPP-A2 and PAPP-A in mice.

Abstract Pregnancy-associated plasma protein-A (PAPP-A) is an important regulatory component of the IGF system. Through proteolysis of inhibitory IGF binding proteins (IGFBPs), PAPP-A acts as a positive modulator of local IGF signaling in a variety of biological systems. A role of IGF in the progression of several common forms of human cancer is now emerging, and therapeutic intervention of IGF receptor signaling is currently being explored. However, little is known about the activities of other components of the IGF system in relation to cancer. We hypothesized that PAPP-A acts to enhance tumor growth in vivo. To test this hypothesis, we overexpressed wild-type PAPP-A or a mutant PAPP-A with markedly reduced IGFBP protease activity in SKOV3 cells, a human ovarian carcinoma cell line with low tumorigenic potential. In vitro, SKOV3 clones with elevated PAPP-A expression (PAPP-A-1, PAPP-A-28) showed accelerated anchorage-independent growth in soft agar assays compared to clones overexpressing mutant PAPP-A (E483Q-1, E483Q-5) and vector controls. PAPP-A-28, with the highest PAPP-A expression and IGFBP proteolytic activity, also had markedly increased cell invasion through Matrigel. In vivo, we found significantly accelerated tumor growth rates of PAPP-A-overexpressing SKOV3 clones compared with mutant PAPP-A and controls. Investigation of angiogenesis indicated that overexpression of PAPP-A favored development of mature tumor vasculature and that tumor precursors of PAPP-A-28 in particular had a significantly higher degree of vascularization months before obvious tumor development. In conclusion, our data show that PAPP-A proteolytic activity enhances the tumorigenic potential of ovarian cancer cells and establish a novel tumor growth-promoting role of PAPP-A.

Pregnancy-associated plasma protein-A (PAPP-A) increases local IGF-I bioavailability through cleavage of inhibitory IGF binding protein (IGFBP)-4 in a variety of systems, including the cardiovascular system. To test the hypothesis that expression of PAPP-A promotes the development of atherosclerotic lesions, we generated transgenic mice that express human PAPP-A in arterial smooth muscle. Four founder lines were characterized for transgenic human PAPP-A mRNA and protein expression, IGFBP-4 protease activity, and tissue specificity. In study I, apolipoprotein E knockout (ApoE KO) mice, a well-characterized mouse model of atherosclerosis, and ApoE KO mice expressing the human PAPP-A transgene at relatively high levels (ApoE KO/Tg) were fed a high-fat diet. At harvest, aortas were dissected and opened longitudinally for en face staining of lipid-rich lesions. Lesion area was increased 3.5-fold in aortas from ApoE KO/Tg compared with ApoE KO mice ( P < 0.001), but no significant difference was seen in lesion number. In study II, replacement of PAPP-A expression in arterial smooth muscle of double ApoE KO/PAPP-A KO mice resulted in a 2.5-fold increase in lesion area ( P = 0.002), without an effect on lesion number. PAPP-A transgene expression was associated with a significant increase in an IGF-responsive gene ( P < 0.001), suggesting increased local IGF-I action. We therefore conclude that expression of human PAPP-A localized to arterial smooth muscle accelerates lesion progression in a mouse model of atherosclerosis. These data provide further evidence for the importance of PAPP-A in the cardiovascular system and suggest PAPP-A as a potential therapeutic target in the control of atherosclerosis.

Abstract Insulin-like growth factor binding protein 4 (IGFBP-4) is secreted by normal human osteoblast-like cells (hOB) and is a potent inhibitor of insulin-like growth factor (IGF) action in vitro. In previous studies, IGF treatment of hOB in culture led to markedly reduced medium levels of IGFBP-4 as detected by western ligand blotting. In the present study, incubation of hOB-conditioned medium (hOB-CM) with IGF under cell-free conditions resulted in a similar loss of IGFBP-4. Both IGF-I and IGF-II were capable of inducing a decrease in IGFBP-4; however, IGF-II was more effective. When the six characterized IGFBP were added to hOB-CM, only IGFBP-4 disappeared in response to IGF-II addition. This IGF-regulated loss of IGFBP-4 was inhibited by metalloproteinase inhibitors and appeared to be due to a proteinase that cleaved IGFBP-4 in 18 and 14 kD fragments identified by western immunoblotting. Conditioned media from eight of eight different donor hOB lines tested exhibited IGFBP-4 proteinase activity. To assess the biologic consequences of IGF-II-induced IGFBP-4 proteolysis, we treated hOB with IGF-II for 5 h, which decreased medium IGFBP-4 by 70%, and then measured IGF-I and insulin stimulation of [3H]thymidine incorporation. IGF-II itself was not mitogenic and had no effect on insulin-stimulated [3H]thymidine incorporation. However, pretreatment of cultured hOB with IGF-II enhanced IGF-I-stimulated [3H]thymidine incorporation threefold. In conclusion, these data indicate that (1) regulation of IGFBP-4 availability can occur via an IGF-dependent, IGFBP-4 proteinase secreted by normal hOB, and (2) IGF-II-induced IGFBP-4 proteolysis is associated with enhanced hOB response to IGF-I. The IGFBP-4/IGFBP-4 proteinase system may be involved in local regulation of IGF action in bone.

Abstract Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method. Trace levels of IGF-II mRNA were observed only in one of three SaOS-2 cell preparations and only by RT-PCR. IGF-II mRNA was absent in MG-63 cells. Thus, our data indicate that (1) normal hOB cells express both IGF-I and IGF-II mRNA and (2) transformation of human osteoblastic cells may alter IGF gene expression. These results provide a basis for selecting appropriate cell models for the study of IGFs in human bone.

Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase that controls the tissue availability of insulin-like growth factor (IGF). Homozygous deletion of PAPPA in mice leads to lifespan extension. Since immune function is an important determinant of individual fitness, we examined the natural immune ecology of PAPPA −/− mice and their wild-type littermates reared under specific pathogen-free condition with aging. Whereas wild-type mice exhibit classic age-dependent thymic atrophy, 18-month-old PAPPA −/− mice maintain discrete thymic cortex and medulla densely populated by CD4 + CD8 + thymocytes that are capable of differentiating into single-positive CD4 and CD8 T cells. Old PAPPA −/− mice have high levels of T cell receptor excision circles, and have bone marrows enriched for subsets of thymus-seeding progenitors. PAPPA −/− mice have an overall larger pool of naive T cells, and also exhibit an age-dependent accumulation of CD44 + CD43 + memory T cells similar to wild-type mice. However, CD43 + T cell subsets of old PAPPA −/− mice have significantly lower prevalence of 1B11 and S7, glycosylation isoforms known to inhibit T cell activation with normal aging. In bioassays of cell activation, splenic T cells of old PAPPA −/− mice have high levels of activation antigens and cytokine production, and also elicit Ig production by autologous B cells at levels equivalent to young wild-type mice. These data suggest an IGF-immune axis of healthy longevity. Controlling the availability of IGF in the thymus by targeted manipulation of PAPPA could be a way to maintain immune homeostasis during postnatal development and aging.

AbstractIGFs are required for normal prenatal and postnatal growth. Although actions of IGFs can be modulated by a family of IGF-binding proteins (IGFBPs) in vitro, these studies have identified a complicated pattern of stimulatory and inhibitory IGFBP effects, so that understanding relevant aspects of IGFBP action in vivo has been limited. Here we have produced a null mutation of one specific IGFBP, IGFBP-4, which is coexpressed with IGF-II early in development. Surprisingly, mutation of IGFBP-4, believed from in vitro studies to be exclusively inhibitory, leads to a prenatal growth deficit that is apparent from the time that the IGF-II growth deficit first arises, which strongly suggests that IGFBP-4 is required for optimal IGF-II-promoted growth during fetal development. Mice encoding a mutant IGFBP-4 protease (pregnancy-associated plasma protein-A), which facilitates IGF-II release from an inactive IGF-II/IGFBP-4 complex in vitro, are even smaller than IGFBP-4 mutant mice. However, the more modest IGFBP-4 growth deficit is completely restored in double IGFBP-4/pregnancy-associated plasma protein-A-deficient mice. Taken together these results indicate not only that IGFBP-4 functions as a local reservoir to optimize IGF-II actions needed for normal embryogenesis, but also establish that IGFBP-4 proteolysis is required to activate most, if not all, IGF-II mediated growth-promoting activity.