Human platelet-derived mitogens. I. Identification of insulinlike growth factors I and II by purification and N alpha amino acid sequence analysis

Human platelet lysates contained potent mitogenic activities for MCF-7 human breast-cancer cells in serum-free-defined media. Because these activities were not replaced by known platelet mitogens, such as platelet-derived growth factor or transforming growth factor beta, we sought to identify the breast cancer cell mitogens by purification and N alpha amino-acid sequencing. Acetic acid extracts of outdated human platelets were concentrated by ammonium sulfate precipitation and fractionated on Sephadex G-50 and Bio-Gel P-10 columns in 0.5 mol/L acetic acid. Two major activities were resolved by molecular sieve methods and fractionated further by reverse-phase high-performance liquid chromatography (HPLC). Purifications (70,000 to 870,000-fold) were accomplished yielding mol wt 7,400 products that were homogeneous as determined by iodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography. The factors were identified as insulinlike growth factor I (IGF-I) and II (IGF-II) and truncated IGF-I by N alpha amino acid microsequencing. In dose-response experiments, platelet-derived IGF-I and IGF-II promoted multiple divisions of the MCF-7 cells with ED50 values of 12 and 100 pg/mL, respectively. The specific activities and other bioassay characteristics of platelet-derived IGF-I and IGF-II were similar to those of recombinant-produced human growth factors. This is the first report of the purification of insulinlike growth factors from human platelet lysates.     

Cytoplasmic transfer of the mitogenic response to platelet-derived growth factor.

BALB/c 3T3 mouse cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA and divide. When cells are treated with PDGF and then fused to untreated cells, the resulting heterokaryons become competent to replicate their DNA. Cytoplasts derived from PDGF-treated cells are also able to transfer the growth response to untreated cells. After cytoplasmic transfer to another cell, the strength of the PDGF-induced mitogenic signal is attenuated by a factor roughly proportional to the increase in total cytoplasmic volume. When RNA synthesis is blocked during PDGF treatment, cells do not acquire the capacity to transfer the PDGF growth signal to untreated cells. By contrast, exposure to cycloheximide during PDGF treatment has no effect. These observations suggest that cytoplasmic transfer of the growth response to PDGF (competence) is mediated by a PDGF-induced stable RNA rather than by PDGF itself or a PDGF--receptor complex. The onset of DNA synthesis in PDGF--control heterokaryons occurs a minimum of 11 hr after cell fusion. Thus the substance that is transferred in these cell fusions is not directly involved in DNA synthesis; rather, it seems to trigger a sequence of events culminating in DNA synthesis.     

Subcellular localization of thrombopoietin in human blood platelets and its release upon thrombin stimulation

Thrombopoietin (TPO) is a major regulator of platelet production. The concentration of circulating TPO seems to be determined by its binding and internalization by megakaryocytes and platelets. To elucidate the platelet compartments involved in TPO metabolism, we investigated intraplatelet TPO by post-embedding immunoelectron microscopy, incubated platelets with recombinant human (rh)TPO coupled to colloidal gold and visualized the TPO uptake using electron microscopy. TPO concentrations were measured in 12 platelet concentrates (PC) before and after stimulation with thrombin and after disruption of platelets by freezing-thawing. In resting platelets, immunogold labelling revealed a prevailing cytoplasmic localization of TPO antigen and minor labelling within the surface-connected canalicular system (SCCS); storage granules were devoid of labelling. In tracer experiments, TPO-gold was observed on the plasma and SCCS membranes and within the cytoplasm. Upon thrombin stimulation, endogenous TPO was still detected within the cytoplasm by immunolabelling, and tracer experiments revealed TPO-gold within the cytoplasm and on fibrin fibres. After thrombin stimulation of PC, the plasma TPO levels increased to an average of 535%, and after platelet lysis to an average of 1625% compared with plasma values in unstimulated PC. We conclude that platelets contain releasable immunoreactive TPO within the SCCS and within their cytoplasm, but not within granular compartments. Stored immunoreactive TPO is released upon thrombin stimulation, but only to a minor degree.     

Human leukemia cells synthesize and secrete proteins related to platelet-derived growth factor.

Human leukemia cells in culture (HL-60) synthesize and secrete proteins that are recognized by antiserum to human platelet-derived growth factor (PDGF). The molecular mass of the intracellular proteins immunoprecipitated by PDGF antiserum ranged from 34 kDa to 240 kDa. PDGF-related proteins were also identified in the conditioned medium of the cells. Several of these immunoprecipitated proteins were glycosylated. A single protein of 46 kDa was immunoprecipitated from the cell-free translation products of mRNA obtained from the leukemia cells. Antiserum to the C but not to the N terminus of the predicted amino acid sequence of the transforming protein p28sis/PDGF-2 also immunoprecipitated proteins secreted by the HL-60 cells. These findings provide a direct demonstration for the synthesis and secretion of PDGF-like proteins by leukemia cells in culture. These proteins do not appear to be coded by the known c-sis/PDGF-2 locus since no sis mRNA was detectable in the HL-60 cells.     

Human bone marrow stromal cell colonies: response to hydrocortisone and dependence on platelet-derived growth factor

Long-term bone marrow cultures are dependent on the formation in vitro of an adherent cell layer that supports hematopoiesis. We have grown bone-marrow-adherent cells, termed stromal colony-forming units, or CFU-ST, as isolated adherent colonies, and examined some of their growth requirements. Bone marrow mononuclear cells separated from aspirates by density centrifugation and cultured in medium supplemented with fetal calf serum or human plasma gave rise to adherent colonies (CFU-ST). An average of 23.4 +/- 2.1 (mean +/- SEM, n = 19) CFU-ST were produced by 10(5) bone marrow mononuclear cells. CFU-ST could not be cultured from similarly prepared peripheral blood mononuclear cells. The colonies were composed of spindle cells, flat cells, and fat-containing cells, with all three types often present in the same colony, suggesting derivation from a common progenitor. Cells were negative for nonspecific esterase and factor VIII antigen. Hydrocortisone added to the cultures at concentrations of 10(-7) M induced the formation of adipose cells in the center of one-third to one-half of the colonies but did not affect CFU-ST number. Human platelet-poor plasma and platelet-rich plasma were substituted for fetal calf serum in the medium. When all determinations for four experiments were averaged, platelet-rich plasma gave 17.8 +/- 1.2 (mean +/- SEM, n = 16) colonies, whereas platelet-poor plasma gave only 0.2 +/- 0.1 colonies (n = 15). When purified platelet-derived growth factor (PDGF) was added to platelet-poor plasma, growth of CFU-ST was enhanced, and a dose-response relationship was found between size of colonies and concentration of added PDGF. Granulocyte-macrophage colony stimulating factor added to cultures had no effect on the growth of CFU-ST.     

Truncated and native insulinlike growth factor I enhance mucosal adaptation after jejunoileal resection.

It has been shown previously that insulinlike growth factors (IGFs) stimulate the proliferation of intestinal crypt cells in vitro. To examine the in vivo effects of IGF-I on mucosal adaptation, three groups of Sprague-Dawley rats underwent 80% jejunoileal resection. Miniosmotic pumps were then inserted under the skin immediately after resection to deliver vehicle (resected control), 1.5 mg/kg per day of IGF-I, or 1.5 mg/kg per day of des-(1-3)-IGF-I (des-IGF-I). Des-IGF-I is a truncated form of IGF-I that binds as well to type I IGF receptors but less tightly to several forms of IGF-binding proteins (IGFBPs) than IGF-I. Ad libitum food intake did not differ among the three resected groups. Body weight gains were greater in animals receiving des-IGF-I than in those receiving IGF-I, which were greater than resected controls. All animals were killed 7 days postoperatively, and the remaining small intestine was removed and divided at the anastomotic site. Both IGF-I and des-IGF-I induced hyperplasia (increased DNA and protein content) in the duodenojejunum but not in the ileum. IGF-I and des-IGF-I were equally active. In contrast, sucrase, maltase, and leucine aminopeptidase activities were greater only in the ileum of animals receiving IGF-I and des-IGF-I than in resected controls. Although more potent in stimulating overall body weight gain, des-IGF-I was not more potent than IGF-I when duodenal and ileal responses were determined. IGF infusion (IGF-I greater than des-IGF-I) increased the levels of circulating IGFBP-3 and IGFBP-2, which may act to modulate the biological effectiveness of the infused peptides. These results suggest that both IGF-I and des-IGF-I may have potential as therapeutic agents for short bowel patients.     

The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor.

We have observed a modification of the cellular protein kinase pp60c-src, elicited in murine 3T3 fibroblasts by platelet-derived growth factor (PDGF). The modification occurred rapidly after addition of PDGF to the culture medium and was first detected as a reduction in the electrophoretic mobility of a portion of the pp60c-src molecules. A similarly modified form of the viral homologue pp60v-src occurs in vivo in the absence of stimulation by PDGF. The occurrence of modified forms of both pp60c-src and pp60v-src was associated with a novel phosphorylation at tyrosine in the amino-terminal domains of the proteins. The time-course and dose-response for this modification of pp60c-src paralleled PDGF-induced increases in phosphorylation of pp36, a major cellular substrate for several tyrosine-specific protein kinases. In parallel experiments, treatment of cells with PDGF increased the kinase activity of pp60c-src in an immunocomplex assay. These results suggest pp60c-src may play a role in the mitogenic response to PDGF.     

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase.

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.     

Bacteria-induced release of white cell- and platelet-derived vascular endothelial growth factor in vitro

BACKGROUND AND OBJECTIVES: Poor prognosis after resection of primary colorectal cancer may be related to the combination of perioperative blood transfusion and subsequent development of infectious complications. White blood cell--and platelet-derived cancer growth substances, including vascular endothelial growth factor (VEGF), may be involved in this process. Therefore, we studied the in vitro release of VEGF from white blood cells and platelets stimulated by bacterial antigens and supernatants from stored red cell components. MATERIALS AND METHODS: Eight units of whole blood (WB) and eight units of buffy-coat-depleted red cell (SAGM) blood were donated by healthy blood donors. Subsequently, half of every unit was leucocyte depleted by filtration, and all 32 half-units were stored under standard conditions for 35 days. Just after storage, and on days 7, 21 and 35 during storage, aliquots of the supernatants were removed from the units and frozen at -80 degrees C. WB from other healthy donors was stimulated for 2 h with sodium chloride (controls), with Escherichia coli lipopolysaccharide (LPS) alone, or with LPS plus supernatants from the non-filtered or prestorage leucofiltered WB units (diluted 1:10), or from non-filtered or prestorage leucofiltered SAGM blood units (diluted 1:20) stored for 0, 7, 21, or 35 days, respectively. Similar assays were performed using Staphylococcus aureus-derived protein A as a stimulatory antigen. The concentration of VEGF was determined in supernatants from stored blood and in assay supernatants by using enzyme-linked immunosorbent assay (ELISA). RESULTS: The concentration of VEGF increased significantly (P < 0.0001) in a storage time-dependent manner in non-filtered WB and SAGM blood, while the increase was abrogated by prestorage leucofiltration. The supernatant concentration of VEGF was significantly increased in LPS-stimulated (P = 0.002) and in protein A-stimulated (P < 0.0001) assays compared with controls. Addition of supernatants from stored, non-filtered WB or SAGM significantly increased the assay supernatant VEGF concentration storage-time dependently (P = 0.006) in LPS assays. In protein A assays, only supernatants from non-filtered WB significantly increased the assay supernatant VEGF concentration storage-time dependently (P = 0.022). This additional effect by supernatants from stored blood components was not observed with prestorage leucofiltered blood. CONCLUSIONS: Extracellular VEGF may accumulate in non-filtered red cell components, but this can be prevented by prestorage leucocyte depletion using filtration. In addition, bacterial antigens appear to induce release of VEGF from white blood cells and platelets. Addition of supernatants from stored, non-filtered WB or SAGM blood may increase the VEGF levels in a storage time-dependent manner, while prestorage leucofiltration may prevent further increase by supernatants.     

Insulin-like growth factor I is essential for postnatal growth in response to growth hormone.

Insulin-like growth factor I (IGF-I) is essential for cell growth and intrauterine development while both IGF-I and GH are required for postnatal growth. To explore the possibility of direct GH action on body growth, independent of IGF-I production, we have studied the effects of GH in an IGF-I-deficient mouse line created by the Cre/loxP system. The IGF-I null mice are born with 35% growth retardation and show delayed onset of peripubertal growth, grow significantly slower, and do not attain puberty. Their adult body weight was approximately one third and body length about two thirds that of their wild-type litter mates. Injection of recombinant human GH (rhGH, 3 mg/kg, twice daily, sc) between postnatal day 14 (P14) to P56 failed to stimulate their growth as measured as both body weight and length. In contrast, wild-type mice receiving the same doses of rhGH exhibited accelerated growth starting at P21 that continued until P56, when their body weight was increased by 30% and length by 12% compared with control mice treated with diluent. Despite the lack of response in growth, IGF-I null mice have normal levels of GH receptor expression in the liver and increased liver Jun B expression and liver size in response to rhGH treatment. Our results support an essential role for IGF-I in GH-induced postnatal body growth in mice.     

Exposure to platelet-derived growth factor modulates the porcine aortic smooth muscle cell response to somatomedin-C

Platelet-derived growth factor (PDGF) is a potent mitogen for smooth muscle cells, but to detect maximal stimulation by PDGF, the cells must be incubated with plasma. Somatomedin-C (Sm-C), a peptide growth factor that is present in plasma, has been shown to interact with PDGF synergistically to stimulate DNA synthesis in cultured fibroblasts. These studies were designed to test the hypothesis that PDGF interacted with Sm-C to stimulate smooth muscle cell replication and to compare the response of this cell type to that of fibroblasts. When PDGF or Sm-C was added individually to smooth muscle cell cultures, each growth factor induced only minimal increases in [3H]thymidine incorporation into DNA (i.e. PDGF, 3,400 +/- 1,120 to 54,900 +/- 1,550 cpm; Sm-C, 3,400 +/- 1,120 to 10,950 +/- 980 cpm). In contrast, addition of increasing concentrations of Sm-C to cultures that were continuously exposed to PDGF in the presence of Sm-C-deficient plasma resulted in a synergistic increase in [3H]thymidine incorporation (3,400 +/- 1,120 to 54,500 +/- 1,800 cpm; P less than 0.001). To determine if Sm-C was required for smooth muscle cell replication, cultures were sequentially exposed to PDGF, followed by Sm-C-deficient plasma. The rate at which the non-Sm-C-exposed cells synthesized DNA was retarded compared to that of cells exposed to Sm-C-containing plasma; however, 68% nuclear labeling was present after 44 h of incubation. To exclude the possibility that some cellular secretory product was substituting for Sm-C, the medium was changed every 2 h and replaced by fresh Sm-C-deficient medium. Using these test conditions, exposure to PDGF and Sm-C-deficient plasma induced only 11% labelling. Readdition of pure Sm-C to this medium restored nuclear labeling to 82% at 44 h. Other variables that appeared to modulate the cellular response to Sm-C were culture density and simultaneous PDGF exposure. Sm-C and PDGF both appear to be potent mitogens for porcine aortic smooth muscle cells, and when added together to quiescent cultures, their effects are synergistic. Smooth muscle cells appear to require Sm-C to initiate DNA synthesis, and in its absence produce a Sm-like factor that can partially compensate for Sm-C deficiency and allow replication.     

Interaction of receptors for insulin-like growth factor I, platelet-derived growth factor, and fibroblast growth factor in rat aortic cells

Serum contains various growth factors which regulate the proliferation of cells. We investigated the growth of cultured arterial smooth muscle cells under the influence of insulin-like growth factor I (IGF I), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and examined the effect of these growth factors on the binding of [125I] IGF I and on the binding of [125I]PDGF to these cells. IGF I, FGF, and PDGF stimulated [6-3H]thymidine incorporation into DNA of confluent cultures of cells which were incubated in modified Dulbecco's modified Eagle medium. However, the effect of these growth factors on DNA synthesis was much more potent in Dulbecco's modified Eagle medium with 1% fetal calf serum. FGF and PDGF potentiated the growth-promoting effect of IGF I. The binding of [125I]IGF I to the cells was increased after a preincubation with FGF and PDGF. The binding was potently increased by FGF (100 ng/ml) after a preincubation time of 30 min. There was an increase in binding during the first 3 h of preincubation followed by a decrease after 4-5 h. PDGF (10-1000 ng/ml) stimulated [125I]IGF I binding only after 2 h of preincubation. The stimulation was dose dependent. Maximal stimulation of the binding was observed after 3 h of preincubation followed by a decrease after 4-5 h of preincubation. Specific binding sites for PDGF on smooth muscle cells could be demonstrated too. A preincubation of confluent cells with IGF I caused a dose-dependent increase in [125I]PDGF binding. These results support the hypothesis that the regulation of the binding of a specific growth factor by a second growth factor is important for the control of cell growth.     

Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.     

血小板源性生长因子及其受体在心血管疾病中的作用

血小板源性生成因子（PDGFs）及其受体（PDGFRs）在心血管疾病中发挥着重要作用,二者不仅参与了动脉粥样硬化（AS）的形成,而且在血管生成、心肌纤维化过程中也扮演了重要角色。PDGFRs被其配体PDGFs激活后,在血管平滑肌细胞、纤维母细胞、内皮细胞等中的表达上调,通过细胞内信号转导通路,促使该类靶细胞增殖分化,从而参与AS、血管生成和心肌纤维化的发生。本文就PDGFs/PDGFRs在心血管疾病中的研究进展做一综述。     

Activation of casein kinase II in response to insulin and to epidermal growth factor.

Insulin treatment enhances casein kinase II (CKII) activity in 3T3-L1 mouse adipocytes and H4-IIE rat hepatoma cells, the magnitude of the activation varying from 30% to 150%. Activation of CKII was apparent after 5 min of exposure of 3T3-L1 cells to insulin, was maximal by 10 min, and persisted through 90 min. The insulin-stimulated activity was inhibited by low concentrations of heparin and was stimulated by spermine. Activation of CKII was effected by physiological concentrations of insulin (EC50 = 0.15 nM), suggesting that the effect is a true insulin response and not one mediated through insulin-like growth factor receptors. Epidermal growth factor (100 ng/ml for 10 min) also activated CKII in A431 human carcinoma cells, which is consistent with other observations that insulin and epidermal growth factor may have some common effects. Insulin stimulation of CKII activity was due to an increase in the maximal velocity of the kinase; the apparent Km for peptide substrate was not altered. Enhanced activity did not appear to result from increased synthesis of CKII protein, because cycloheximide did not block the effect and because an immunoblot developed with antiserum to CKII showed no effect of insulin on the cytosolic concentration of CKII. Because insulin-stimulated CKII activity was maintained after chromatography of cell extracts on Sephadex G-25, it is unlikely that the effect is mediated by a low-molecular-weight activator of the kinase. Rather, the results are consistent with the possibility that insulin activates CKII by promoting a covalent modification of the kinase.     

Subcellular localization and secretion of factor V from human platelets.

Factor V, a plasma protein cofactor necessary for optimal conversion of prothrombin to thrombin, is also present in considerable concentration in blood platelets (9.9 units per 10(9) platelets). Subcellular fractionation by two methods has localized factor V in the alpha granules of unstimulated platelets. ADP and epinephrine cause release of 4.6% and 6.4%, respectively, of the total factor V, a process completely inhibited by cyclooxygenase alkylation by aspirin. In contrast, collagen causes release of 25% of platelet factor V, a process only partially suppressed by aspirin. Secretion of factor V depends on the availability of metabolic energy, because antimycin A, an inhibitor of aerobic metabolism, and 2-deoxyglucose, an inhibitor of anaerobic glycolysis, together almost totally inhibited the secretion of factor V induced by collagen. The data establish that factor V is not normally available on unstimulated platelets but can be secreted from alpha granules upon stimulation with physiological agents such as ADP, epinephrine, and collagen. Because factor V is known to serve as a receptor for factor Xa, the exposure of factor V on platelets consequent to release would accelerate the process of blood coagulation.     

Transforming growth factor beta induces myoblast differentiation in the presence of mitogens.

Transforming growth factor beta 1 (TGF-beta 1) added to L6E9 rat skeletal myoblasts in mitogen-rich medium induces a rapid decrease in c-myc expression and delays progression through the G1 phase of the cell cycle. This growth inhibitory response is followed by cell commitment to terminal differentiation with elevated expression of myogenin muscle determination genes, induction of muscle-specific proteins, and formation of multinucleated myotubes. These results suggest that TGF-beta 1 may act as a physiological inducer of myogenic differentiation in mitogen-rich environments, and its otherwise reversible growth inhibitory effect may become permanent if coupled to induction of terminal differentiation.     

Prognostic value of insulinlike growth factor I and its binding protein in patients with alcohol-induced liver disease

Insulinlike growth factor I (IGF-I) is a single-polypeptide chain with important anabolic and endocrine activities. The liver is the major source of IGF-I and its binding protein, IGFBP-3. Circulating concentrations of IGF-I and IGFBP-3 are decreased in patients with chronic liver disease and correlate with the severity. The aim of this study was to assess the additional prognostic value of IGF-I and IGFBP-3 in patients entered in a large multicenter study (EMALD). Three hundred thirty-seven patients with alcohol-induced liver disease were studied in a randomized placebo-controlled trial of malotilate with a mean follow-up period of 569 days (range, 7-1,544). A multivariate Cox regression analysis of pertinent clinical and biochemical variables showed a significant independent prognostic value of years of alcohol intake, coagulation factors 2, 7, and 10, alkaline phosphatases, serum creatinine, and immunoglobulin (Ig) M. When IGF-I or IGFBP-3 were added into this model, a Cox regression analysis showed that either had a significant independent prognostic value. Because IGF-I and IGFBP-3 were closely correlated, they contained almost the same prognostic information. Inclusion of IGF-I gave these results: IGF-I (P < .03), alcohol intake (P < .02), coagulation factors 2, 7, and 10 (P < .01), creatinine (P < .001), and IgM (P < .01) contained independent prognostic information. Inclusion of IGFBP-3 gave these results: IGFBP-3 (P < .02), alcohol intake (P < .05), coagulation factors 2, 7, 10 (P < .01), creatinine (P < .001), and IgM (P < .02) were independent predictors of survival. In conclusion, IGF-I or IGFBP-3 provide important additional information on survival in patients with alcohol-induced liver disease. (Hepatology 1996 May;23(5):1073-8)     

The role of alpha and beta platelet-derived growth factor receptor in the vascular response to injury in nonhuman primates

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.