TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

<div><p>Background</p><p>RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.</p><p>Methods and Results</p><p>TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105^−/− mice resulting in a higher proliferation rate. In RP105^−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm² vs.1947±278 µm²,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105^−/− mice this effect was more pronounced (10316±1243 µm² vs.4208±555 µm²,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm²,p<0.05), whereas expression of the single factors RP105 or MD1 had no effect.</p><p>Conclusion</p><p>RP105 is a potent inhibitor of post-interventional neointima formation.</p></div

Neointima formation in WT and RP105^−/− mice.

<p>Neointima formation after femoral artery cuff placement in RP105^−/− and wild type mice. Areas of femoral arterial sections were quantified by using 6 sequential sections per segment and are expressed in micrometers squared (mean±SEM). Increased neointima formation in RP105^−/− mice compared to WT (wild type) controls (A). Increased intima/media ratio in RP105^−/− mice compared to WT controls (B). Representative pictures of Elastin von Giesson (C) HPS (D) and α-smooth muscle cell actin (F) of RP105^−/− and WT controls. * = P<0.05 Arrows indicate the Internal Elastic Lamina (IEL) and the External Elastic Lamina (EEL).</p

Soluble RP105 protein – functionality.

<p>Whole blood stimulation. Blood stimulated with LPS and PBS or combined with purified solRP105 protein or purified solRP105-MD1 protein. Supernatant from triplicates was used for TNFα measurement. * = P<0.05.</p

Soluble RP105 protein – characterization.

<p>Purification and analysis of RP105, MD1 and RP105-MD1 complex. RP105 (lane 1) and RP105-MD1 complex (lane 2) purified by nickel- and streptactin- tandem affinity purification and analysed by SDS-PAGE (10%) and subsequent silverstaining. Lane 3 shows MD1 purified by nickel-affinity purification and analysed by SDS-PAGE (12%) and subsequent Coomassie staining (A). SDS-PAGE analysis of PNGase deglycosylation of SDS denatured MD1 (B). Size exclusion chromatography (Superdex200 PC3.2/30) of the purified RP105 (dashed line) and RP105-MD1 complex (solid line). Molecular weight determination of the RP105-MD1 complex using SEC-MALS (inset). The dashed horizontal lines indicate a molecular weight of 180 kD and 220 kD, respectively (C).</p

SolRP105-MD1 reduced neointima formation.

<p>Neointima formation after femoral artery cuff placement with LPS in hypercholesterolemic APOE3Leiden mice. Areas of femoral arterial sections were quantified by using 6 sequential sections per segment and are expressed in micrometers squared (mean±SEM). Decreased neointima formation in mice that had overexpression of solRP105-MD1 (A). Intima Media ratio (B) Pictures of Elastin von Giesson (C) HPS (D) α-smooth muscle cell actin (E) and CD45 (F) of Luc(1) solRP105 (2) MD1 (3) solRP105-MD1 (4) * = P<0.05.</p

LPS induced neointima formation.

<p>Neointima formation after femoral artery cuff placement with LPS in RP105^−/− and wild type mice. Areas of femoral arterial sections were quantified by using 6 sequential sections per segment and are expressed in micrometers squared (mean±SEM). Increased neointima formation in RP105^−/− mice compared to WT controls after local LPS application (A). Increased intima/media ratio in RP105^−/− mice compared to WT controls after local LPS application (B). Increased outward remodeling in RP105^−/− mice compared to WT controls after local LPS application (C) Representative pictures of Elastin von Giesson (D), HPS (E) and α-smooth muscle cell actin (F) of RP105^−/− and WT controls. * = P<0.05 Arrows indicate the Internal Elastic Lamina (IEL) and the External Elastic Lamina (EEL).</p

RT-PCR of TLR4 (A) or RP105 (B) on wild type VSMC that were incubated with PBS, 1 ng/ml LPS or 10 ng/ml LPS.

<p>Immunostaining of TLR4 (green) or RP105 (red) or both (overlay) on wild type VSMC that were incubated with PBS (C) or 1 ng/ml LPS. (D) Colocalized TLR4 and RP105 staining on VSMC (E). RP105 staining in media area of a murine femoral artery (F). Proliferation of VSMC of WT and RP105^−/− (G) Liquid-scintillation counting after 16 hours of WT and RP105^−/− VSMC. VSMC of show a RP105^−/−an increased proliferative response to LPS. VSMC were cultured in medium containing 10%FCS or 10%FCS with1 ng/ml LPS. Starved WT and RP105^−/− VSMC were used as controls. White bars represent WT VSMC, black bars represent RP105^−/− VSMC.</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

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