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Enhanced platelet activation mediates the accelerated angiogenic switch in mice lacking histidine-rich glycoprotein.

Ringvall M, Thulin Å, Zhang L, Cedervall J, Tsuchida-Straeten N, Jahnen-Dechent W, Siegbahn A, Olsson AK - PLoS ONE (2011)

Bottom Line: Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times.To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα.Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: The heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice.

Methodology/principal findings: To address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG(-/-) mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG(+/+) littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice.

Conclusions: Our data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.

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HRG regulates platelet levels in the circulation.(A) Platelet numbers in whole blood from RT2 positive or negative mice with different HRG genotypes (HRG+/+ or HRG−/−). (B) Left panel; Quantification of the number of platelets/field in kidney tissue from healthy (RT2-negative) HRG+/+ or HRG−/− mice. Right panel; Quantification of the area percentage stained positive for the platelet marker CD41 in tumor tissue from RT2-positive HRG+/+ and HRG−/− mice. (C, D) Immunohistochemical staining directed against CD41 was performed on kidney from RT2-negative mice (C) and tumor tissue from RT2-positive mice (D) from both genotypes. Arrows indicate platelets. (E) HRG+/+ and HRG−/− mice were treated with Plavix (25 mg/ml or 50 mg/ml in drinking water) for three days. Platelet activation in whole blood was measured after stimulation with 10 µM ADP by flow cytometric analysis of the activated fibrinogen receptor (GPIIb/IIIa). (D) Platelet levels after Plavix treatment were measured by flow cytometric analysis in whole blood and are presented as the percentage of GPIX positive cells. Statistical analyses were performed with a two-tailed Mann-Whitney test, vertical bars represent standard deviation, * p≤0.05, ns = non significant. Scale bars represent 50 µm in C and 100 µm in D.
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pone-0014526-g005: HRG regulates platelet levels in the circulation.(A) Platelet numbers in whole blood from RT2 positive or negative mice with different HRG genotypes (HRG+/+ or HRG−/−). (B) Left panel; Quantification of the number of platelets/field in kidney tissue from healthy (RT2-negative) HRG+/+ or HRG−/− mice. Right panel; Quantification of the area percentage stained positive for the platelet marker CD41 in tumor tissue from RT2-positive HRG+/+ and HRG−/− mice. (C, D) Immunohistochemical staining directed against CD41 was performed on kidney from RT2-negative mice (C) and tumor tissue from RT2-positive mice (D) from both genotypes. Arrows indicate platelets. (E) HRG+/+ and HRG−/− mice were treated with Plavix (25 mg/ml or 50 mg/ml in drinking water) for three days. Platelet activation in whole blood was measured after stimulation with 10 µM ADP by flow cytometric analysis of the activated fibrinogen receptor (GPIIb/IIIa). (D) Platelet levels after Plavix treatment were measured by flow cytometric analysis in whole blood and are presented as the percentage of GPIX positive cells. Statistical analyses were performed with a two-tailed Mann-Whitney test, vertical bars represent standard deviation, * p≤0.05, ns = non significant. Scale bars represent 50 µm in C and 100 µm in D.

Mentions: To further analyze platelet activation in vivo in HRG knockout mice, we measured the level of circulating platelets as well as the presence of platelets in tissue from HRG+/+ and HRG−/− mice. Activated platelets leave the circulation and become arrested in the vascular bed. A reduced number of platelets in the blood can therefore reflect an increase in platelet activation. We analyzed the number of circulating platelets in whole blood from healthy (RT2-negative) wild type and HRG-deficient mice using a cell counter. There was a significant decrease in the number of platelets in blood from HRG−/− mice compared to wild type (Fig. 5A), supporting the conclusion that platelets are more easily activated in the absence of HRG. It is well established that tumors can promote coagulation and platelet activation, for instance by expression of tissue factor [22]. In agreement, platelet levels were significantly reduced in blood from wild type mice with insulinoma (RT2/HRG+/+) compared to healthy wild types (HRG+/+) (Fig. 5A). However, RT2-positive HRG−/− mice (RT2/HRG−/−) showed no further decrease in platelet numbers compared to HRG−/− mice without tumors (HRG−/−). Healthy HRG-deficient mice had in fact similar platelet levels as wild type mice with insulinoma (Fig. 5A).


Enhanced platelet activation mediates the accelerated angiogenic switch in mice lacking histidine-rich glycoprotein.

Ringvall M, Thulin Å, Zhang L, Cedervall J, Tsuchida-Straeten N, Jahnen-Dechent W, Siegbahn A, Olsson AK - PLoS ONE (2011)

HRG regulates platelet levels in the circulation.(A) Platelet numbers in whole blood from RT2 positive or negative mice with different HRG genotypes (HRG+/+ or HRG−/−). (B) Left panel; Quantification of the number of platelets/field in kidney tissue from healthy (RT2-negative) HRG+/+ or HRG−/− mice. Right panel; Quantification of the area percentage stained positive for the platelet marker CD41 in tumor tissue from RT2-positive HRG+/+ and HRG−/− mice. (C, D) Immunohistochemical staining directed against CD41 was performed on kidney from RT2-negative mice (C) and tumor tissue from RT2-positive mice (D) from both genotypes. Arrows indicate platelets. (E) HRG+/+ and HRG−/− mice were treated with Plavix (25 mg/ml or 50 mg/ml in drinking water) for three days. Platelet activation in whole blood was measured after stimulation with 10 µM ADP by flow cytometric analysis of the activated fibrinogen receptor (GPIIb/IIIa). (D) Platelet levels after Plavix treatment were measured by flow cytometric analysis in whole blood and are presented as the percentage of GPIX positive cells. Statistical analyses were performed with a two-tailed Mann-Whitney test, vertical bars represent standard deviation, * p≤0.05, ns = non significant. Scale bars represent 50 µm in C and 100 µm in D.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3022027&req=5

pone-0014526-g005: HRG regulates platelet levels in the circulation.(A) Platelet numbers in whole blood from RT2 positive or negative mice with different HRG genotypes (HRG+/+ or HRG−/−). (B) Left panel; Quantification of the number of platelets/field in kidney tissue from healthy (RT2-negative) HRG+/+ or HRG−/− mice. Right panel; Quantification of the area percentage stained positive for the platelet marker CD41 in tumor tissue from RT2-positive HRG+/+ and HRG−/− mice. (C, D) Immunohistochemical staining directed against CD41 was performed on kidney from RT2-negative mice (C) and tumor tissue from RT2-positive mice (D) from both genotypes. Arrows indicate platelets. (E) HRG+/+ and HRG−/− mice were treated with Plavix (25 mg/ml or 50 mg/ml in drinking water) for three days. Platelet activation in whole blood was measured after stimulation with 10 µM ADP by flow cytometric analysis of the activated fibrinogen receptor (GPIIb/IIIa). (D) Platelet levels after Plavix treatment were measured by flow cytometric analysis in whole blood and are presented as the percentage of GPIX positive cells. Statistical analyses were performed with a two-tailed Mann-Whitney test, vertical bars represent standard deviation, * p≤0.05, ns = non significant. Scale bars represent 50 µm in C and 100 µm in D.
Mentions: To further analyze platelet activation in vivo in HRG knockout mice, we measured the level of circulating platelets as well as the presence of platelets in tissue from HRG+/+ and HRG−/− mice. Activated platelets leave the circulation and become arrested in the vascular bed. A reduced number of platelets in the blood can therefore reflect an increase in platelet activation. We analyzed the number of circulating platelets in whole blood from healthy (RT2-negative) wild type and HRG-deficient mice using a cell counter. There was a significant decrease in the number of platelets in blood from HRG−/− mice compared to wild type (Fig. 5A), supporting the conclusion that platelets are more easily activated in the absence of HRG. It is well established that tumors can promote coagulation and platelet activation, for instance by expression of tissue factor [22]. In agreement, platelet levels were significantly reduced in blood from wild type mice with insulinoma (RT2/HRG+/+) compared to healthy wild types (HRG+/+) (Fig. 5A). However, RT2-positive HRG−/− mice (RT2/HRG−/−) showed no further decrease in platelet numbers compared to HRG−/− mice without tumors (HRG−/−). Healthy HRG-deficient mice had in fact similar platelet levels as wild type mice with insulinoma (Fig. 5A).

Bottom Line: Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times.To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα.Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: The heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice.

Methodology/principal findings: To address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG(-/-) mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG(+/+) littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice.

Conclusions: Our data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.

Show MeSH
Related in: MedlinePlus