Rapamycin as a potent and selective inhibitor of vascular endothelial growth factor receptor in breast carcinoma.

Angiogenesis is the process of new vascular formation, which is derived from various factors. For suppressing cancer cell growth, targeting angiogenesis is one of the therapeutic approaches. Vascular endothelial growth factor family receptors, including Flt-1, Flk-1 and Flt-4, have been found to play an essential role in regulating angiogenesis. Rapamycin is a macrolide compound with anti-proliferative properties, while platelet factor-4 (PF-4) is an antiangiogenic ELR-negative chemokine. Rapamycin inhibits mTOR ligands activation, thus suppressing cell proliferation, while PF-4 inhibits cell proliferation through several mechanisms. In the present study, we evaluated the effects of rapamycin and platelet factor-4 toward breast carcinoma at the proteomic and genomic levels. A total of 60 N-Methyl-N-Nitrosourea-induced rat breast carcinomas were treated with rapamycin, platelet factor-4 and rapamycin+platelet factor-4. The tumours were subsequently subjected to immunohistochemical protein analysis and polymerase chain reaction gene analysis. Protein analysis was performed using a semiquantitative scoring method, while the mRNA expression levels were analysed based on the relative expression ratio. There was a significant difference in the protein and mRNA expression levels for the selected markers. In the rapamycin+platelet factor-4-treated group, the Flt-4 marker was downregulated, whereas there were no differences in the expression levels of other markers, such as Flt-1 and Flk-1. On the other hand, platelet factor-4 did not exhibit a superior angiogenic inhibiting ability in this study. Rapamycin is a potent antiangiogenic drug; however, platelet factor-4 proved to be a less effective drug of anti-angiogenesis on rat breast carcinoma model.

CD8+ T cells inhibit metastasis and CXCL4 regulates its function.

BACKGROUND: The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. METHODS: We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. RESULTS: We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. CONCLUSIONS: CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.

The cytokine platelet factor 4 successfully replaces bovine serum albumin for the in vitro culture of porcine embryos.

The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100-1000 ng/mL, 0 = control) to NCSU-23 with 0.4 mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100-1000 ng/mL, 0 = Control-PVA) to a BSA-free medium (NCSU-23 with 0.3 mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4 mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0 ±â€¯10.9 to 70.0 ±â€¯5.8%); of day 7-blastocyst rates (range: 46.6 ±â€¯10.0 to 56.4 ±â€¯8.2%) and of total day 7-blastocyst efficiency (range: 32.3 ±â€¯8.3 to 37.2 ±â€¯7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1 ±â€¯23.2 to 50.5 ±â€¯26.4). In Experiment 2, PF4 accelerated embryo development, increasing (P < 0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100-500 ng/mL (range: 29.9 ±â€¯7.8 to 31.8 ±â€¯5.5%; P < 0.05) compared with BSA-control (17.2 ±â€¯8.2%) and PF4 1000 ng/mL (15.5 ±â€¯7.9%); showing similar blastocyst rates (range: 42.0 ±â€¯11.5 to 49.3 ±â€¯10.0%), total efficiency (28.0 ±â€¯8.2 to 32.3 ±â€¯7.1%) total cell numbers (range: 42.6 ±â€¯19.3 to 45.7 ±â€¯23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions.

Exogenous CXCL4 infusion inhibits macrophage phagocytosis by limiting CD36 signalling to enhance post-myocardial infarction cardiac dilation and mortality.

Aims: Macrophage phagocytosis of dead cells is a prerequisite for inflammation resolution. Because CXCL4 induces macrophage phagocytosis in vitro, we examined the impact of exogenous CXCL4 infusion on cardiac wound healing and macrophage phagocytosis following myocardial infarction (MI). Methods and results: CXCL4 expression significantly increased in the infarct region beginning at Day 3 post-MI, and macrophages were the predominant source. Adult male C57BL/6J mice were subjected to coronary artery occlusion, and MI mice were randomly infused with recombinant mouse CXCL4 or saline beginning at 24 h post-MI by mini-pump infusion. Compared with saline controls, CXCL4 infusion dramatically reduced 7 day post-MI survival [10% (3/30) for CXCL4 vs. 47% (7/15) for saline, P < 0.05] as a result of acute congestive heart failure. By echocardiography, CXCL4 significantly increased left ventricular (LV) volumes and dimensions at Day 5 post-MI (all P < 0.05), despite similar infarct areas compared with saline controls. While macrophage numbers were similar at Day 5 post-MI, CXCL4 infusion increased Ccr4 and Itgb4 and decreased Adamts8 gene levels in the infarct region, all of which linked to CXCL4-mediated cardiac dilation. Isolated Day 5 post-MI macrophages exhibited comparable levels of M1 and M4 markers between saline and CXCL4 groups. Interestingly, by both ex vivo and in vitro phagocytosis assays, CXCL4 reduced macrophage phagocytic capacity, which was connected to decreased levels of the phagocytosis receptor CD36. In vitro, a CD36 neutralizing antibody (CD36Ab) significantly inhibited macrophage phagocytic capacity. The combination of CXCL4 and CD36Ab did not have an additive effect, indicating that CXCL4 regulated phagocytosis through CD36 signalling. CXCL4 infusion significantly elevated infarct matrix metalloproteinase (MMP)-9 levels at Day 5 post-MI, and MMP-9 can cleave CD36 as a down-regulation mechanism. Conclusion: CXCL4 infusion impaired macrophage phagocytic capacity by reducing CD36 levels through MMP-9 dependent and independent signalling, leading to higher mortality and LV dilation.

Cellular and Molecular Changes in MNU-Induced Breast Tumours Injected with PF4 or bFGF

Background: Angiogenic activity has been considered to reflect important molecular events during breast tumour development. The present study concerned cellular and molecular changes of MNU-induced breast tumours subjected to promotion and suppression of angiogenesis. Methods: Female Sprague Dawley rats at the age of 21 days received MNU at the dose 70 mg/kg of body weight by intraperitoneal injection. Three months post-carcinogen initiation, mammary tumours were palpated and their growth was monitored. When the tumour diameter reached 1.0 ± 0.05 cm, rats were given bFGF or PF4 intratumourally at a dose of 10 µg/tumour. Entire palpable tumour were subsequently excised and subjected to histology examination, IHC staining, and RT-PCR. Results: No critical morphological changes were observed between pro-angiogenic factor, bFGF, and control groups. However, increase of tumour size with more necrotic and diffuse areas was notable in tumours after anti-angiogenic PF4 intervention. ER and PR mRNA expression was significantly up- and down-regulated in bFGF and PF4 groups, respectively. The trends were significantly associated with peri- and intratumoural MVD counts. However, irrespective of whether we promoted or inhibited angiogenesis, the expression of EGFR and ERBB2 continued to be significantly increased but this was not significantly associated with the MVD score. No significant differences in E-cadherin and LR gene expression were noted between intervention and control groups. Conclusion: ER and PR receptor expression shows consistent responses when tumour angiogenesis is manipulated either positively or negatively. Our study adds to current understanding that not only do we need to target hormonal receptors, as presently practiced, but we also need to target endothelial receptors to successfully treat breast cancer.

5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies.

Essentials No humanized monoclonal antibody was available to study heparin-induced thrombocytopenia (HIT). We developed the first anti-platelet factor 4 (PF4)/heparin antibody with a human Fc fragment. This antibody (5B9) fully mimics the effects of human HIT antibodies. 5B9 binds two regions within PF4 that may be critical for the pathogenicity of HIT antibodies. SUMMARY: Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.

PF4/heparin complexes are T cell-dependent antigens.

Heparin-induced thrombocytopenia (HIT) is a life-threatening, thrombotic disorder associated with development of anti-platelet factor 4 (anti-PF4)/heparin autoantibodies. Little is known about the antigenic and cellular requirements that initiate the immune response to these complexes. To begin to delineate mechanisms of autoantibody formation in HIT, we studied the immunizing effects of murine PF4 (mPF4)/heparin in mice with and without thymic function. Euthymic mice were injected with mPF4/heparin complexes, mPF4, heparin, or buffer. Mice injected with mPF4/heparin, but not mPF4 or heparin alone, developed heparin-dependent autoantibodies that shared serologic and functional characteristics of human HIT antibodies, including preferential binding to mPF4/heparin complexes and causing heparin- and FcRgammaIIA-dependent platelet activation. In contrast, athymic mice did not develop HIT-like antibodies. Taken together, these studies establish that PF4/heparin complexes are highly immunogenic and elicit self-reacting anti-PF4/heparin antibodies in a T cell-dependent manner.

Local intracerebral delivery of endogenous inhibitors by osmotic minipumps effectively suppresses glioma growth in vivo.

The systemic administration of endogenous inhibitors significantly reduced the growth of human glioma in vivo, but required the production of a large amount of biologically active protein. In this study we reduced the amount of protein needed and optimized the therapeutical response by delivering the endogenous inhibitors locally into the brain by osmotic minipumps. Human hemopexin fragment of MMP-2 or COOH-terminal fragment of platelet factor-4 were delivered locally and continuously into the brain of mice implanted intracranially with glioma cells, by osmotic minipumps connected to an intracranial catheter. Local delivery of human hemopexin fragment of MMP-2 and COOH-terminal fragment of platelet factor-4 significantly inhibited the growth of well-established malignant glioma in nude and BALB/C mice. When the inhibitors were given at the same concentration, the efficacy of the local delivery was much higher than that reached with the systemic administration, both when the inhibitor was administered daily or continuously by s.c. minipumps. Moreover, the local delivery reduced the amount of protein needed to reach a significant therapeutic response. Intracerebral delivery maintained a long-term control of glioma growth and inhibited glioma recurrence in a surgical resection model. Treatment showed no side effects. Histochemical analysis of tumors showed that the tumor growth inhibition was the result of a decrease in tumor vasculature and a change in tumor vessel morphology. Our data demonstrate that local intracerebral delivery of endogenous inhibitors effectively inhibits malignant glioma growth and reduces the amount of protein needed to reach a therapeutical response.

Recombinant platelet factor 4, an angiogenic marker for human breast carcinoma.

The present study was designed to test the hypothesis that rhPF4 binds with high specificity to the neovasculature of breast cancer carcinoma. To achieve this goal, we used intravital microscopy to study the binding characteristics of systemically injected fluorescently labeled rhPF4 (FITC-rhPF4) to the microvasculature of dorsal skinfold chambers in nude mice implanted with tumor spheroids prepared from the human breast cancer cell line MCF-7. Our results show that intravenously as well as intra-arterially injected FITC-rhPF4 exclusively labeled, with high intensity and specificity, the endothelium of the breast cancer induced neovasculature. Only on rare occasions (0.7 +/- 1.5 site per cm2 skinfold), short (37 +/- 48 microns) intense labeled sites were found in the normal vasculature of the skinfold. Heparin could displace most of the label if injected within 10 min after the rhPF4-injection, but not 30 min after. In conclusion, our results show that rhPF4 preferentially binds to regions of active angiogenesis in vivo, supporting the concept of using rhPF4 conjugates to target tumors in cancer patients. Certain rhPF4 conjugates could have applications as imaging agents, with potential utility in identification, screening, detection, prognosis or staging of breast cancer and other cancers. Other similar conjugates, bearing therapeutic isotopes or toxins might be useful in selective treatment strategies.

A novel biological effect of platelet factor 4 (PF4): enhancement of LPS-induced tissue factor activity in monocytes.

In a previous study we have shown that granulocytes enhance lipopolysaccharide (LPS)-induced tissue factor (TF) activity in monocytes in a platelet-dependent reaction. The present investigation was undertaken to examine the role of a platelet activation product, platelet factor 4 (PF4), in LPS-induced TF activity in monocytes. Platelet lysate supernatant, purified PF4, and the COOH-terminal tridecapeptide of PF4, termed PF4(58-70), enhanced LPS-induced TF activity in monocytes of whole blood dose dependently. A monoclonal antibody against P-selectin eliminated the enhancing effect of PF4(58-70) on LPS-induced TF activity in monocytes, and PF4(58-70) was shown to act synergistically with tumor necrosis factor alpha (TNF-alpha). However, PF4(58-70) did not enhance TNF-alpha secretion in LPS-stimulated whole blood. The major effect of PF4(58-70) was granulocyte dependent. Our results suggest that PF4 might play an important role in LPS-stimulated monocyte TF activity of whole blood.

Heparin neutralization by recombinant platelet factor 4 and protamine.

Protamine is the only available drug to reverse heparin-induced anticoagulation. Platelet factor 4 (PF4) is a basic polypeptide stored in platelets that reverses heparin. To investigate its potential as a reversal drug, we studied recombinant PF4 on anticoagulated blood obtained during cardiac surgery. Blood was obtained from 33 different venous reservoirs, and activated clotting time (ACT), heparin concentrations, and heparinase-ACT were determined. Anticoagulation was reversed by adding incremental PF4:heparin and protamine:heparin ratios to the heparinized blood, and the ACTs were determined (n = 21). Viscoelastic analysis of anticoagulation reversal was performed by adding protamine or PF4 at reversal ratios of 1.3:1 protamine:heparin, and 3.2:1 PF4:heparin using thromboelastography (n = 12). PF4 reversal ratios of 3:1 and 3.5:1 and protamine reversal ratios of 1:1, 1.5:1, 2:1 were not statistically different from heparinase-ACT values. There were no significant differences in viscoelastic measurements of clot formation between protamine and PF4. Recombinant PF4 at a 3.0:1 ratio reverses heparin-induced anticoagulation after cardiopulmonary bypass, and represents a potential alternative, especially for the protamine allergic patient.

Hepatic arterial infusion of recombinant platelet factor-4 suppresses metastases to the lungs from tumors implanted into the livers of rabbits.

BACKGROUND: This study evaluated the toxicity (Part I) and antitumor effects (Part II) associated with hepatic arterial infusion of recombinant platelet factor-4 (rPF4), an antiangiogenic protein. METHODS: Healthy rabbits (Part I) and rabbits with tumors implanted in their livers (Part II) received saline or rPF4 via hepatic arterial infusion. Three saline-receiving and four rPF4-receiving animals died 2-3 days postinfusion from gastroduodenal thromboembolism. The remaining animals were necropsied 3, 7, 10, or 14 days postinfusion. Blood analyses and hepatic angiography were performed before infusion and at the time of sacrifice. RESULTS: In Part I, focal coagulation necrosis of the hepatic parenchyma was observed in 1 of 11 rabbits that received saline and in 6 of 10 that received rPF4. In Part II, hepatic arterial infusion of rPF4 had no effect on growth of the implanted liver tumors. However, the protein significantly reduced the incidence of lung metastasis. CONCLUSIONS: Intraarterial infusion of rPF4 significantly reduced the incidence of lung metastasis. Nonheparin systemic anticoagulation may be needed during catheterization and infusion procedures to prevent thromboemboli.

Reversal of heparin anticoagulation by recombinant platelet factor 4 in humans.

BACKGROUND: Protamine is used to reverse the anticoagulant effects of heparin, but it can have important side effects. Platelet factor 4 (PF4) is a protein found in platelet alpha granules that binds to and thereby neutralizes heparin. We evaluated the safety and effectiveness of intravenous recombinant PF4 to neutralize heparin anticoagulation after cardiac catheterization in a phase 1, open-label trial. METHODS AND RESULTS: The study group consisted of 18 patients having diagnostic cardiac catheterization. Heparin (5000 U) was given after vascular access was obtained. In the first 12 patients, additional heparin was given at the conclusion of the procedure so that all patients had activated coagulation times > 300 seconds before rPF4 was given. Three patients each received 0.5, 1.0, 2.5, or 5.0 mg/kg rPF4 over a period of 3 minutes at the conclusion of the catheterization procedure. In 6 additional patients, extra heparin was not given at the conclusion of the procedure, and 1.0 mg/kg rPF4 was given. Hemodynamic measurements, cardiac output, and serial blood tests were performed 5, 10, 20, and 30 minutes after rPF4 and then into the next 24 hours. There were no serious side effects in any patient, despite transient rPF4 levels as high as 14,870 ng/mL in the patients receiving 5.0 mg/kg. One patient receiving 2.5 mg/kg had a slight transient rise in liver enzymes possibly related to the rPF4. There were no important hemodynamic effects of rPF4 administration at any dose used. Doses of 2.5 and 5.0 mg/kg were uniformly effective in reversing the anticoagulant effect of heparin. At lower doses, rPF4 neutralized the effects of heparin in most but not all patients. Pharmacokinetic analysis suggested a monophasic and one-compartment clearance of the PF4-heparin complex. No neutralizing factors to rPF4 were detected in the samples collected 7 days after dosing. CONCLUSIONS: rPF4, in doses ranging from 0.5 to 5.0 mg/kg over 3 minutes, had no serious side effects. Given in sufficient amounts, rPF4 can completely and rapidly reverse the anticoagulant effects of heparin.

Inhibition of development of murine melanoma lung metastases by systemic administration of recombinant platelet factor 4.

BACKGROUND: When administered locally, recombinant platelet factor 4 (rPF4), a known angiogenesis inhibitor, has been shown to effectively suppress murine melanoma and human colon carcinoma primary tumor growth in mice. It was tentatively concluded that this effect was due to the inhibition of tumor neovascularization. PURPOSE: This study has evaluated the effects of systemically administered rPF4 on the growth and establishment of experimental B16F10 melanoma lung metastases in syngeneic mice. METHODS: B16F10 cells (0.5-1.0 x 10(5) were administered intravenously to mice; 21 days later, the lungs were removed and the tumor foci were counted. Treatments with rPF4 were given to C57BL/6J mice immediately following tumor inoculation either (a) intravenously as a single dose (0.375, 0.75, 1.5, or 2.0 mg) or as multiple doses (6 mg total) over a 48-hour period, (b) subcutaneously or intramuscularly as multiple doses (6 mg total) over a 72-hour period, or (c) subcutaneously as multiple doses (6 mg total) over a 92- or 96-hour period following a delay of 4, 24, or 48 hours. (BALB/cByJ x C57BL/6)F1 (CByB6F1/J) athymic nude mice received rPF4 subcutaneously over a 72-hour period. The ability of rPF4 to block binding of 51Cr-labeled B16F10 tumor cells to matrix-coated microtiter plates was evaluated in vitro. The in vivo effect of intravenously injected rPF4 on the retention of 51Cr-labeled B16F10 cells was examined by determining the remaining lung-associated radioactivity after 30 minutes or 1, 2, or 4 hours. RESULTS: Intravenous administration of rPF4 significantly inhibited the development of metastatic lung nodules in a dose-dependent fashion as assessed by both lesion number (P < .03) and lung weight (P < .05). When initiation of subcutaneous treatment with rPF4 was delayed 24-48 hours, the number of metastatic foci was significantly reduced (P < .05). The antitumor effect of rPF4 was not dependent on a T-lymphocyte-mediated process, since subcutaneous rPF4 treatment also suppressed the number of lung metastases in T-cell-deficient athymic CByB6F1/J nude mice. In vitro experiments demonstrated modest inhibitory effects (28%) of rPF4 on B16F10 tumor cell binding to purified murine vitronectin. However, lung clearance experiments at early time points (0.5 hour and 1 hour) showed that tumor lodging was not affected by rPF4 treatment. CONCLUSIONS: The administration of rPF4 by systemic routes inhibited the development of experimental lung metastases. These findings are consistent with the known angiostatic properties of rPF4, and we conclude that inhibition of metastatic tumor formation may be due to inhibition of tumor-induced neovascularization. IMPLICATIONS: These results support the testing of rPF4 as an angiostatic agent in the treatment of metastatic tumors.

Platelet factor 4 injection produces acute pulmonary hypertension in the awake lamb.

BACKGROUND: Reversal of heparin anticoagulation by intravenous protamine sulfate consistently produces acute pulmonary vasoconstriction mediated by the release of thromboxane in the awake lamb. Recently, recombinant platelet factor 4 (rPF4) has been cloned, expressed in Escherichia coli, and infused to reverse heparin anticoagulation in the rat, without producing adverse hemodynamic or pulmonary morphologic effects. The authors sought to learn whether intravenous administration of PF4 is devoid of side effects in the pulmonary circulation of lambs. METHODS: The authors evaluated the hemodynamic response and plasma release rates of thromboxane during intravenous challenges with heparin-rPF4 (n = 2), rPF-free carrier (n = 5), rPF4 (n = 5), rPF4 after indomethacin (n = 5), protamine (n = 5) and heparin-protamine (n = 5) in 17 awake, hemodynamically monitored lambs. Each lamb underwent up to three random challenges with a 2-h recovery period between each challenge. RESULTS: In two lambs, systemic anticoagulation with heparin followed by reversal of anticoagulation with an intravenous bolus of rPF4 (4 mg/kg) led to acute pulmonary vasoconstriction and hypertension with the release of thromboxane (peak pulmonary artery pressure [Ppa] 40 and 33 mmHg and peak plasma thromboxane B2 50 and 30 ng/ml, respectively). Intravenous administration of rPF4 (1.5 mg/kg) alone increased the Ppa from 17.2 +/- 0.7 mmHg (mean +/- SEM) at baseline to 31.2 +/- 2 mmHg at 1 min (n = 5, P < 0.05). This was associated with an increase of plasma thromboxane B2 from 0.06 +/- 0.02 to 3.96 +/- 1.21 ng/ml. Acute pulmonary vasoconstriction lasted approximately 5 min and was completely prevented by pre-treatment with oral indomethacin (10 mg/kg). Intravenous bolus administration of rPF4 carrier (n = 5) or protamine (2 mg/kg) alone (n = 5) did not induce pulmonary hypertension or the release of thromboxane. In five lambs, intravenous heparin (200 U/kg) followed by protamine (2 mg/kg) consistently produced acute pulmonary vasoconstriction and hypertension. CONCLUSIONS: Intravenous injection of human rPF4 into the awake lamb produces acute pulmonary vasoconstriction and hypertension associated with thromboxane release into circulating blood. The effects of rPF4 on the pulmonary vasculature should be evaluated in primates before rPF4 is substituted for protamine in reversing heparin anticoagulation in humans.

Chemokines/intercrines and central regulation of feeding.

Chemokines/intercrines are structurally and functionally related cytokines that induce specific actions on the immune system and are released in response to infection, inflammation, and trauma. These pathological processes are frequently accompanied with food intake suppression. In the present study, the action of chemokines/intercrines on the regulation of feeding was investigated using the intracerebroventricular microinfusion of chemokine/intercrine-alpha subfamily members [interleukin-8 (IL-8); growth-related cytokine/melanoma growth-stimulating activity (GRO-alpha/MGSA); platelet factor-4 (PF-4); beta-thromboglobulin (beta-TG); and interferon-inducible protein-10 (IP-10)] and beta-subfamily members [monocyte chemotactic protein-1/monocyte chemotactic and activating factor (MCP-1/MCAF); regulated upon activation normal T-cell expressed and presumably secreted (RANTES); macrophage inflammatory protein-1 alpha (MIP-1 alpha); and macrophage inflammatory protein-1 beta (MIP-1 beta)]. The doses administered were 1.0, 20, and 100 ng/rat of the chemokine/intercrine. The intracerebroventricular administration of three members of the alpha-subfamily (IL-8, PF-4, and IP-10) and two members of the beta-subfamily (MCP-1/MCAF and RANTES) decreased the short-term (2-h) food intake. These effective chemokines/intercrines, however, were significantly less potent than IL-1 beta in decreasing feeding. The results support the hypothesis that only a subset of immunomodulators released during pathological processes may participate in the regulation of feeding with different potencies.

Food intake suppression by growth factors and platelet peptides by direct action in the central nervous system.

Intracerebroventricular (i.c.v.) microinfusion of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and platelet factor 4 (PF4) suppressed 2 h and nighttime food intake in rats. The following daytime food intake did not change or increased. I.c.v. infusion of platelet-derived growth factor (PDGF) suppressed only 2 h food intake. I.c.v. infusion of bovine serum albumin (BSA), nerve growth factor (NGF), or inactivated EGF, bFGF and PF4 had no effect. Intraperitoneal (i.p.) administration of EGF, bFGF, PF4 and PDGF in doses equivalent to or higher than those administered centrally had no effect. The results suggest a central action of growth factors and platelet peptides on feeding regulation.