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Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo.

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells.Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

ABSTRACT
Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

No MeSH data available.


Related in: MedlinePlus

AngII infusion causes a CC-chemokine dependent recruitment of monocytes to the aorta that is inhibited by R89A 35 K-Fc.Mice were treated with AngII at 1 mg/kg/day by subcutaneous osmotic minipump. Aorta were harvested from mice following 3 days of infusion and RNA was prepared. A significant induction of CCL2 was measured in the mice following AngII infusion compared to baseline uninfused control animals (n = 4) (A). Digestion of isolated aortas showed CCR2+/CD11b+ leukocytes within the aorta (B) these cells were significantly increased in number following 7 days of Ang II infusion, vs saline control animals (n = 4–8) (C). Aortas harvested over a timecourse of AngII infusion showed a progressive recruitment of leuckoytes and in particular monocytes and macrophages to the aorta (n = 5–8) (D). To assess the efficacy of R89A 35K-Fc in inhibiting this recruitment, mice were injected with 1 μg R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to implanatation of an AngII minipump. Mice were sacrificed after 72 hours of AngII treatment (E). Analysis of plasma confirmed expression of 35 K-Fc by ELISA (F). Analysis of the recruited cells showed a significant blunting of the recruitment of total CD45+ leukocytes, CD11b+ myeloid cells and monocytes (inflammatory 7/4HI and total 7/4HI/INT) to the aorta, but no significant effect on neutrophil recruitment (G) (n = 12–13 AngII, n = 4 Saline). *p < 0.05. Statistical testing was performed by T-test (two groups only) or One-way ANOVA with Dunnett’s post testing.
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f4: AngII infusion causes a CC-chemokine dependent recruitment of monocytes to the aorta that is inhibited by R89A 35 K-Fc.Mice were treated with AngII at 1 mg/kg/day by subcutaneous osmotic minipump. Aorta were harvested from mice following 3 days of infusion and RNA was prepared. A significant induction of CCL2 was measured in the mice following AngII infusion compared to baseline uninfused control animals (n = 4) (A). Digestion of isolated aortas showed CCR2+/CD11b+ leukocytes within the aorta (B) these cells were significantly increased in number following 7 days of Ang II infusion, vs saline control animals (n = 4–8) (C). Aortas harvested over a timecourse of AngII infusion showed a progressive recruitment of leuckoytes and in particular monocytes and macrophages to the aorta (n = 5–8) (D). To assess the efficacy of R89A 35K-Fc in inhibiting this recruitment, mice were injected with 1 μg R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to implanatation of an AngII minipump. Mice were sacrificed after 72 hours of AngII treatment (E). Analysis of plasma confirmed expression of 35 K-Fc by ELISA (F). Analysis of the recruited cells showed a significant blunting of the recruitment of total CD45+ leukocytes, CD11b+ myeloid cells and monocytes (inflammatory 7/4HI and total 7/4HI/INT) to the aorta, but no significant effect on neutrophil recruitment (G) (n = 12–13 AngII, n = 4 Saline). *p < 0.05. Statistical testing was performed by T-test (two groups only) or One-way ANOVA with Dunnett’s post testing.

Mentions: Having shown that R89A 35 K-Fc protein expression following hydrodynamic delivery was systemically available and effective in a model of peritoneal inflammation we next decided to test whether the protein would be effective in another, more complex pathology that has been reported to be CC chemokine dependent15. We infused C57bl/6J animals with Angiotensin II (Ang II) at 1 mg/kg/day and performed gene expression studies on the aorta from mice 3 after 3 days of AngII infusion compared to un-infused controls. We showed a significant induction of aortic CCL2 expression following Ang II treatment (Fig. 4A). To assess whether this expression results in an increased recruitment of leukocytes bearing the CCL2 receptor, CCR2, we isolated aortas from untreated and Ang II infused mice. We performed enzymatic digests and used flow cytometry and a cell counting protocol to enumerate the number of CCR2+ cells in the aorta (Fig. 4B). In keeping with the increased expression of CCL2 we observed that the number of CCR2+ myeloid (CD11b+) cells resident in the aorta 7 days after initiation of AngII infusion was increased (Fig. 4C). We next quantified the recruitment of leukocytes (defined as CD45+) in response to AngII infusion in more detail. Leukocytes were recruited to the aorta over the 7 days of the experiment, with a significant recruitment of monocytes within 3 days, a number that remained unchanged at 7 days (Fig. 4D). In contrast only small numbers of cells exhibiting macrophage markers were recruited to the aorta within 3 days, but this number was significantly increased at 7 days, presumably as the recruited monocytes differentiated to become macrophages.


Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo.

McNeill E, Iqbal AJ, White GE, Patel J, Greaves DR, Channon KM - Sci Rep (2015)

AngII infusion causes a CC-chemokine dependent recruitment of monocytes to the aorta that is inhibited by R89A 35 K-Fc.Mice were treated with AngII at 1 mg/kg/day by subcutaneous osmotic minipump. Aorta were harvested from mice following 3 days of infusion and RNA was prepared. A significant induction of CCL2 was measured in the mice following AngII infusion compared to baseline uninfused control animals (n = 4) (A). Digestion of isolated aortas showed CCR2+/CD11b+ leukocytes within the aorta (B) these cells were significantly increased in number following 7 days of Ang II infusion, vs saline control animals (n = 4–8) (C). Aortas harvested over a timecourse of AngII infusion showed a progressive recruitment of leuckoytes and in particular monocytes and macrophages to the aorta (n = 5–8) (D). To assess the efficacy of R89A 35K-Fc in inhibiting this recruitment, mice were injected with 1 μg R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to implanatation of an AngII minipump. Mice were sacrificed after 72 hours of AngII treatment (E). Analysis of plasma confirmed expression of 35 K-Fc by ELISA (F). Analysis of the recruited cells showed a significant blunting of the recruitment of total CD45+ leukocytes, CD11b+ myeloid cells and monocytes (inflammatory 7/4HI and total 7/4HI/INT) to the aorta, but no significant effect on neutrophil recruitment (G) (n = 12–13 AngII, n = 4 Saline). *p < 0.05. Statistical testing was performed by T-test (two groups only) or One-way ANOVA with Dunnett’s post testing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664965&req=5

f4: AngII infusion causes a CC-chemokine dependent recruitment of monocytes to the aorta that is inhibited by R89A 35 K-Fc.Mice were treated with AngII at 1 mg/kg/day by subcutaneous osmotic minipump. Aorta were harvested from mice following 3 days of infusion and RNA was prepared. A significant induction of CCL2 was measured in the mice following AngII infusion compared to baseline uninfused control animals (n = 4) (A). Digestion of isolated aortas showed CCR2+/CD11b+ leukocytes within the aorta (B) these cells were significantly increased in number following 7 days of Ang II infusion, vs saline control animals (n = 4–8) (C). Aortas harvested over a timecourse of AngII infusion showed a progressive recruitment of leuckoytes and in particular monocytes and macrophages to the aorta (n = 5–8) (D). To assess the efficacy of R89A 35K-Fc in inhibiting this recruitment, mice were injected with 1 μg R89A 35 K-Fc plasmid or GFP control plasmid 5 days prior to implanatation of an AngII minipump. Mice were sacrificed after 72 hours of AngII treatment (E). Analysis of plasma confirmed expression of 35 K-Fc by ELISA (F). Analysis of the recruited cells showed a significant blunting of the recruitment of total CD45+ leukocytes, CD11b+ myeloid cells and monocytes (inflammatory 7/4HI and total 7/4HI/INT) to the aorta, but no significant effect on neutrophil recruitment (G) (n = 12–13 AngII, n = 4 Saline). *p < 0.05. Statistical testing was performed by T-test (two groups only) or One-way ANOVA with Dunnett’s post testing.
Mentions: Having shown that R89A 35 K-Fc protein expression following hydrodynamic delivery was systemically available and effective in a model of peritoneal inflammation we next decided to test whether the protein would be effective in another, more complex pathology that has been reported to be CC chemokine dependent15. We infused C57bl/6J animals with Angiotensin II (Ang II) at 1 mg/kg/day and performed gene expression studies on the aorta from mice 3 after 3 days of AngII infusion compared to un-infused controls. We showed a significant induction of aortic CCL2 expression following Ang II treatment (Fig. 4A). To assess whether this expression results in an increased recruitment of leukocytes bearing the CCL2 receptor, CCR2, we isolated aortas from untreated and Ang II infused mice. We performed enzymatic digests and used flow cytometry and a cell counting protocol to enumerate the number of CCR2+ cells in the aorta (Fig. 4B). In keeping with the increased expression of CCL2 we observed that the number of CCR2+ myeloid (CD11b+) cells resident in the aorta 7 days after initiation of AngII infusion was increased (Fig. 4C). We next quantified the recruitment of leukocytes (defined as CD45+) in response to AngII infusion in more detail. Leukocytes were recruited to the aorta over the 7 days of the experiment, with a significant recruitment of monocytes within 3 days, a number that remained unchanged at 7 days (Fig. 4D). In contrast only small numbers of cells exhibiting macrophage markers were recruited to the aorta within 3 days, but this number was significantly increased at 7 days, presumably as the recruited monocytes differentiated to become macrophages.

Bottom Line: We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis.Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells.Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Medicine, British Heart Foundation Centre for Research Excellence, John Radcliffe Hospital , University of Oxford, U.K.

ABSTRACT
Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.

No MeSH data available.


Related in: MedlinePlus