CCR7-CCL19/CCL21 Axis is Essential for Effective Arteriogenesis in a Murine Model of Hindlimb Ischemia

Craipeau Maria. Le conformiste Metello Les clowns Les faucons Five Easy Pieces Performance. In: Raison présente, n°19, Juillet – Août – Septembre 1971. Télévision, pouvoir et liberté. pp. 115-116

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

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

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

Recruitment of Ly6C^hi monocytes in RP105^−/− mice in vivo.

<p>Flow cytometry analysis of monocytes subtypes (Ly6C^hi and Ly6^l°) before (t0) and 1 day after (t1) HLI in WT and RP105^−/− mice (n = 6 WT t0; n = 6 WT t1; n = 6 RP105^−/− t0; n = 6 RP105^−/− t1). Activation state of Ly6C^hi monocytes measured by mean fluorescence intensity (MFI) of CD11b in blood (<b>A</b>) and ischemic gastrocnemius muscle (<b>B</b>). Flow cytometry analysis of the Ly6C^hi monocyte population in adductor muscle group (<b>C</b>), gastrocnemius muscle (<b>D</b>), bone marrow (<b>E</b>), spleen (<b>F</b>) and blood (<b>G</b>). Flow cytometry analysis of the Ly6C^l° monocyte population in adductor muscle group (<b>H</b>), gastrocnemius muscle (<b>I</b>), bone marrow (<b>J</b>), spleen (<b>K</b>) and blood (<b>L</b>). <b>M</b>, WT and RP105^−/− bone marrow-derived monocyte migration with and without LPS stimulation (10 ng/ml) (n = 4). *P<0.05, **P<0.01, ***P<0.001.</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

RP105 and TLR4 in arteriogenesis in WT mice.

<p>RP105 mRNA (<b>A</b>) and TLR4 mRNA (<b>B</b>) expression in the adductor muscle group after induction of HLI, measured by real-time quantitative PCR. RP105 mRNA (<b>C</b>) and TLR4 mRNA (<b>D</b>) expression in the ischemic gastrocnemius muscle after induction of HLI, measured by real-time quantitative PCR (n = 4 mice per time point). Expression levels were normalized against either GAPDH or RPL13A. pt = pre-treatment. All values are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, calculated against pre-treatment. Immunohistochemical staining on paraffin-embedded WT adductor muscle group (<b>E</b>) and gastrocnemius muscle (<b>F</b>) 1 day after induction of HLI, using anti-RP105 antibodies. Black arrowheads denote RP105⁺ cells. (<b>G</b>) Immunohistochemical staining on fresh-frozen sections of WT adductor muscle 1 day after induction of HLI, using anti-αSMA (red), anti-RP105 (white) and anti-MOMA-2 (green) antibodies. Cell nuclei were stained with DAPI (blue). White arrowheads denote RP105⁺MOMA-2⁺ cells.</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