Effect of garcinol treatment on inflammatory cell recruitment and CCL2 expression <i>in vivo</i>.

<p>(A) Quantification of CD45 positive cells (leukocytes) in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 8). *<i>P</i><0.05, **<i>P</i><0.01. (B) Quantification of Mac3 positive cells (macrophages) in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 6). **<i>P</i><0.01. (C) Quantification of CCL2 positive cells in the intima and media 3 days after cuff placement in ApoE*3-Leiden mice treated with garcinol (n = 6) or pluronic gel (n = 7). **<i>P</i><0.01, ***<i>P</i><0.001. Representative images of CD45, Mac3 and CCL2 staining of cuffed femoral arteries, scale bar = 20 μm. Results are mean±SEM.</p

TLR4 Accessory Molecule RP105 (CD180) Regulates Monocyte-Driven Arteriogenesis in a Murine Hind Limb Ischemia Model

<div><p>Aims</p><p>We investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6C^hi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105⁺ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia.</p><p>Methods and Results</p><p>RP105^−/− and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105^−/− mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6C^hi monocytes were more readily activated in RP105^−/− mice. FACS analyses showed that Ly6C^hi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6C^hi monocytes were readily activated in RP105^−/− mice, migration into the ischemic tissues was hampered and instead, Ly6C^hi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105^−/− mice.</p><p>Conclusions</p><p>RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6C^hi monocytes results in reduced infiltration of Ly6C^hi monocytes in ischemic tissues and in impaired blood flow recovery.</p></div

Effect of PCAF deficiency on intimal hyperplasia and vascular smooth muscle cell content <i>in vivo</i>.

<p>Representative images of PCAF staining (A), scale bar = 100 μm. Quantification of intimal hyperplasia (B), intima/media ratio (C) and lumenstenosis (D) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 11) mice. ***<i>P</i><0.001. Representative images of elastin staining (E), scale bar = 50 μm. Quantification intimal (F) and medial (G) smooth muscle cell area (μm²) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 8) mice. **<i>P</i><0.001. (H) Representative images of smooth muscle actin (SMA) staining of cuffed femoral arteries, scale bar = 50 μm. Results are mean±SEM.</p

Blood flow recovery in RP105^−/− mice.

<p>(<b>A</b>) Representative Laser Doppler Perfusion Imaging (LDPI) images of paws from WT and RP105^−/− mice after induction of HLI in the left limb. High blood flow is displayed in red. (<b>B</b>) Quantification of LDPI measurements of RP105^−/− (n = 10) and WT (n = 9) mice over time. Data are calculated as the ratio of ligated over non-ligated paw. (<b>C</b>) Quantification of LDPI measurements of WT and RP105^−/− mice directly after induction of HLI. (<b>D</b>) Quantification of LDPI measurements 10 days after induction of HLI. (<b>E</b>) Immunohistochemical staining of paraffin-embedded adductor muscle group of WT (n = 6) and RP105^−/− (n = 6) mice, 10 days after HLI, using anti-αSMA (red) antibodies. Smallest lumen diameter of αSMA⁺ vessels is indicated by black bars. (<b>F</b>) Immunohistochemical staining on fresh frozen sections of gastrocnemius muscles of WT (n = 6) and RP105^−/− (n = 6) mice, 10 days after HLI, using anti-CD31 (brown) antibodies. Number (<b>G</b>) and lumen area (µm²) (<b>H</b>) of αSMA⁺ vessels, measured at the center of the adductor muscle group in ligated and non-ligated limbs of RP105^−/− and WT mice. (<b>I</b>) Capillary density in gastrocnemius muscles, defined as the number of CD31⁺ vessels per section. pt = pre-treatment. ns = non-significant. All values are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.</p

Effect of PCAF deficiency on inflammatory cytokine expression <i>in vitro</i>.

<p>(A) TNF-alpha production of whole blood derived leukocytes (n = 5) from WT and PCAF KO mice 24 hours after LPS (0–250 ng/ml) stimulation. *<i>P</i><0.05. TNF-alpha(B) and IL-6 (C) production of bone marrow derived macrophages (n = 3) from WT and PCAF KO mice 24 hours after LPS (0–250 ng/ml) stimulation. *<i>P</i><0.05. (D) CCL2 production of vascular smooth muscle cells (n = 3) from WT and PCAF KO mice 24 hours after LPS (0–1 ng/ml) stimulation. *<i>P</i><0.05, ***<i>P</i><0.001. Results are mean±SEM.</p

Effect of PCAF deficiency on macrophage influx and CCL2 expression <i>in vivo</i>.

<p>Quantification of Mac3 positive cells (macrophages) in the intima (A) and media (B) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 11) mice. Representative images of Mac3 staining (C), scale bar = 50 μm. Quantification of CCL2 positive area in the intima (D) and media (E) 21 days after cuff placement in WT (n = 7) and PCAF KO (n = 11) mice. Representative images of CCL2 staining (F), scale bar = 50 μm. Results are mean±SEM.</p

Effect of garcinol treatment and PCAF downregulation on inflammatory cytokine expression <i>in vitro</i>.

<p>(A) TNF-alpha production of whole blood derived leukocytes (n = 5) after 24 hours stimulation with LPS (100 ng/ml) in combination with garcinol (0–20 μM). *<i>P</i><0.05 compared to vehicle (0 μM garcinol), n.d.: non-detectable. (B) CCL2 production of vascular smooth muscle cells (n = 3) after 24 hours stimulation with LPS (1 ng/ml) in combination with garcinol (0–15 μM). *<i>P</i><0.05 compared to vehicle (0 μM garcinol). (C) Whole blood derived leukocyte viability (n = 4) after 24 hours incubation with garcinol (0–250 μM), expressed as percentage fluorescence intensity. ***<i>P</i><0.001 compared to 0 μM garcinol. (D) Relative Pcaf expression of vascular smooth muscle cells (n = 3) transfected with scrambled or PCAF siRNA and stimulated with 1 ng/ml for 24 hours. **<i>P</i><0.01 compared to scrambled siRNA. (E) CCL2 production of vascular smooth muscle cells (n = 3) transfected with scrambled or PCAF siRNA and stimulated with 1 ng/ml for 24 hours. *<i>P</i><0.05 compared to scrambled siRNA. Results are mean±SEM.</p

Monocyte activation in RP105^−/− mice after induction of HLI.

<p>LDPI quantification of WT (<b>A</b>) and RP105^−/− (<b>B</b>) mice. Mice were injected with LPS (1 µg/mouse) in PBS (n = 11 WT; n = 11 RP105^−/−) or PBS alone (n = 9 WT; n = 9 RP105^−/−) at 3 days after induction of HLI. Data are calculated as the ratio of ligated over non-ligated paw. All values are presented as the mean ± SEM. Ctrl = control. ***P<0.001.</p

Pre-existing collateral bed in RP105^−/− mice.

<p>(<b>A</b>) Representative images of the pial circulation in WT and RP105^−/− mice. White asterisks indicate collateral arteries between anterior, middle and posterior cerebral arteries (ACA, MCA and PCA, respectively). (<b>B</b>) Pial collateral density was calculated in WT (n = 4) and RP105^−/− (n = 4) mice, dividing the sum of ACA to MCA, ACA to PCA and MCA to PCA connectors by the surface area of the cerebral hemispheres. (<b>C</b>) Region of the brain utilized for calculation of pial density. Areas were excluded when they were damaged, had poor filling with Microfil, or were otherwise uncountable. ns = non-significant. All values are presented as the mean ± SEM.</p

Inflammatory response in RP105^−/− mice.

<p>Blood from WT and RP105^−/− mice was collected, diluted (1∶25) and incubated for 24 h with LPS (0–75 ng) ex vivo. TNFα (pg/ml) (<b>A</b>) and IL6 (pg/ml) (<b>B</b>) levels in cell-free supernatant were measured by ELISA (n = 5 WT; n = 5 RP105^−/−). Plasma TNFα levels (pg/ml) (<b>C</b>) and SAA1 (µg/ml) (<b>D</b>) in RP105^−/− and WT mice, 1 h after intraperitoneal injection of LPS (1 µg/mouse) (n = 8 WT PBS; n = 9 WT LPS; n = 9 RP105^−/− PBS; n = 10 RP105^−/− LPS). ST2L mRNA (<b>E</b>) and SIGIRR mRNA (<b>F</b>) expression in the adductor muscle group 10 days after induction of HLI, measured by real-time quantitative PCR (n = 6 WT; n = 6 RP105^−/−). (<b>G</b>) Flow cytometry analysis of monocytes and monocyte subtypes (Ly6C^hi and Ly6C^l°) in RP105^−/− and WT mice. Values are presented as total counts in blood (n x10⁶/mL). Fraction of Ly6C^hi (<b>H</b>) and Ly6^l° (<b>I</b>) subtypes of total monocytes in RP105^−/− and WT mice after incubation with LPS or control ex vivo. Activation state of total monocytes (<b>J</b>), Ly6C^hi monocytes (<b>K</b>) and Ly6^l° monocytes (<b>L</b>) in whole blood incubated with LPS or control ex vivo, measured by mean fluorescence intensity (MFI) of CD11b (n = 5 WT PBS; n = 5 WT LPS; n = 5 RP105^−/− PBS; n = 5 RP105^−/− LPS). nd = non-detectable, ctrl = control. All values are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.</p