VEGFR1-Positive Macrophages Facilitate Liver Repair and Sinusoidal Reconstruction after Hepatic Ischemia/Reperfusion Injury

<div><p>Liver repair after acute liver injury is characterized by hepatocyte proliferation, removal of necrotic tissue, and restoration of hepatocellular and hepatic microvascular architecture. Macrophage recruitment is essential for liver tissue repair and recovery from injury; however, the underlying mechanisms are unclear. Signaling through vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to play a role in macrophage migration and angiogenesis. The aim of the present study was to examine the role of VEGFR1 in liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion (I/R). VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK^-/- mice) and wild-type (WT) mice were subjected to hepatic warm I/R, and the processes of liver repair and sinusoidal reconstruction were examined. Compared with WT mice, VEGFR1 TK^-/- mice exhibited delayed liver repair after hepatic I/R. VEGFR1-expressing macrophages recruited to the injured liver showed reduced expression of epidermal growth factor (EGF). VEGFR1 TK^-/- mice also showed evidence of sustained sinusoidal functional and structural damage, and reduced expression of pro-angiogenic factors. Treatment of VEGFR1 TK^-/- mice with EGF attenuated hepatoceullar and sinusoidal injury during hepatic I/R. VEGFR1 TK^-/- bone marrow (BM) chimeric mice showed impaired liver repair and sinusoidal reconstruction, and reduced recruitment of VEGFR1-expressing macrophages to the injured liver. VEGFR1-macrophages recruited to the liver during hepatic I/R contribute to liver repair and sinusoidal reconstruction. VEGFR1 activation is a potential therapeutic strategy for promoting liver repair and sinusoidal restoration after acute liver injury.</p></div

Hepatic expression of growth factors and angiogenic factors in WT and VEGFR1 TK^-/- mice after hepatic I/R.

<p>(A–D) IL-6 (A), TNFα (B), HGF (C), and EGF (D) mRNA levels in livers from WT mice and VEGFR1 TK^-/- mice measured by real-time PCR. Data are expressed as the mean ± SEM from six mice per group. *p<0.05 <i>vs</i>. WT mice. (E–G) Levels of Ang1 (E), Ang2 (F), and Tie2 (G) mRNA in livers from WT mice and VEGFR1 TK^-/- mice. Data are expressed as the mean ± SEM from six mice per group. *p<0.05 <i>vs</i>. WT mice. (H) Double staining of Tie2 and Lyve-1 at 48 h. Tie2 (red) and Lyve-1 (green) colocalize in the sinusoids. Cell nuclei are stained by DAPI (blue). Scale bar, 50 µm.</p

Effect of EGF on liver injury after hepatic I/R.

<p>(A) Double immunostaining of mouse livers with antibodies against EGF (red) and VEGFR1 (green) at 48 h post-reperfusion. Scale bar, 50 µm. (B) EGF-positive cells in WT and VEGFR1 TK^-/- livers. Data are expressed as the mean ± SEM from five to six mice per group. *p<0.05 <i>vs</i>. WT mice. (C,D) Effects of EGF or PBS on liver injury in VEGFR1 TK^-/- mice at 48 h post-reperfusion. (C) ALT levels, necrotic area, hemorrhagic area, and PCNA index. (D) Levels of Ang1, Ang2, and Tie2 mRNA in the liver at 48 h post-reperfusion. Data are expressed as the mean ± SEM from five to six mice per group. p<0.05 <i>vs</i>. PBS-treated WT mice.</p

Delayed liver repair and sinusoidal reconstruction after hepatic I/R in VEGFR1 TK^-/- mice.

<p>Changes in VEGF-A (A), VEGFR1 (B), and VEGFR2 (C) mRNA levels in livers from WT mice and VEGFR1 TK^-/- mice after hepatic I/R, and changes in ALT levels (D), the area of hepatic necrosis (E), the PCNA index (F), and the hemorrhagic area (G). Representative <i>in vivo</i> microscopic images showing the uptake of acetylated LDL (white dots) at 24 h and 48 h (H). PP, periportal region; CL, centrilobular region. Sinusoidal perfusion after hepatic I/R (I). Data are expressed as the mean ± SEM from five to six mice per group. *p<0.05 <i>vs</i>. WT mice.</p

Recruitment of VEGFR1-positive cells from the BM, and hepatic I/R injury in BM chimeric mice.

<p>BM cells from GFP+WT mice (WT→WT) or GFP+VEGFR1 TK^-/- mice (VEGFR1 TK^-/- →WT) were injected into the tail vein of WT mice. (A,B) VEGFR1-positive cells (A) and double-positive cells (GFP- and VEGFR1-positive) (B) in the liver at 48 h post-reperfusion. (C) Expression of VEGFR1 and CD11b in the peripheral blood. Representative flow cytometry plots for GFP+WT BM (left panel) and GFP+VEGFR1 TK^-/- BM chimeric mice (right panel) are shown. (D) The percentage of BM-derived VEGFR1/CD11b-positive cells at 48 h at 48 h post-reperfusion.as assessed by FACS analysis. (E) Effect of transplanting BM cells from WT mice and VEGFR1 TK-/- mice on liver repair and hepatocyte proliferation at 48 h post-reperfusion. ALT levels, the area of hepatic necrosis, the hemorrhagic area, and the PCNA index were determined. Data are expressed as the mean ± SEM from six mice per group. *p<0.05 <i>vs</i>. GFP+WT BM chimeric mice.</p

<i>CDO1</i> methylation and expression in BC cell line.

<p><b>A,</b><i>CDO1</i> mRNA expression in BC cell lines was assessed by semi-quantitative reverse transcribed PCR (RT-PCR). B, Representative direct bisulfite sequence results in CRL cells (methylation) and SK-BR3 cells (unmethylation). C, <i>CDO1</i> mRNA expression in BC cell lines was assessed by Q-MSP. (D) <i>CDO1</i> mRNA expression in BC tissues was assessed by RT-PCR.</p

<i>CDO1</i> gene methylation and prognosis according to BC subtypes.

<p>A, Kaplan-Meier curve for DSS is shown in total primary BC. The cut-off value was median value (58). Patients with <i>CDO1</i> hypermethylation exhibited significantly poorer prognosis than those with <i>CDO1</i> hypomethylation in primary BC (p = 0.004). B, Kaplan-Meier curves for DSS are shown according to subtypes.</p

Quantitative assessment of <i>CDO1</i> methylation in primary BC tissues.

<p><b>A,</b> TaqMeth value of 172 BC tissues.Median is median 58.0 (0–351.1). B, p value and relative risk were plotted according to the Log rank test. Note that the p value is constantly below 0.05, suggesting that the higher methylation value of <i>CDO1</i> gene is, poorer prognosis the patients exhibited in primary BC.</p

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BackgroundCD44 and CD133 are stem cell markers in colorectal cancer (CRC). CD44 has distinctive isoforms with different oncological properties like total CD44 (CD44T) and variant CD44 (CD44V). Clinical significance of such markers remains elusive.MethodsSixty colon cancer were examined for CD44T/CD44V and CD133 at mRNA level in a quantitative PCR, and clarified for their association with clinicopathological factors.Results(1) Both CD44T and CD44V showed higher expression in primary colon tumors than in non-cancerous mucosas (pCD133 was expressed even in non-cancerous mucosa and rather decreased in the tumors (p = 0.048). (2) CD44V expression was significantly associated with CD44T expression (R = 0.62, pCD133 at all in the primary tumors. (3) CD44V/CD44T expressions were significantly higher in right colon cancer than in left colon cancer (p = 0.035/p = 0.012, respectively), while CD133 expression were not (p = 0.20). (4) In primary tumors, unexpectedly, CD44V/CD44T/CD133 mRNA expressions were not correlated with aggressive phenotypes, but CD44V/CD44T rather significantly with less aggressive lymph node metastasis/distant metastasis (p = 0.040/p = 0.039, respectively). Moreover, both CD44V and CD133 expressions were significantly decreased in liver metastasis as compared to primary tumors (p = 0.0005 and p = 0.0006, respectively).ConclusionOur transcript expression analysis of cancer stem cell markers did not conclude that their expression could represent aggressive phenotypes of primary and metastatic tumors, and rather represented less demand on stem cell marker-positive cancer cells.</div

Clinical significance of CD133 expression in primary colon cancer tissues.

(a) There was no significant difference in the expression of CD133 between right and left primary colon cancer in non cancerous mucosa tissues (p = 0.34). And similar results were obtained for N factors and M factors (p = 0.58 and p = 0.12, respectively). (b) As well as (a), there was no significant difference in the expression of CD133 between right and left primary colon cancer in colon cancer tumor tissues (p = 0.20) as well as N factors and M factors(p = 0.12 and p = 0.28, respectively). (PDF)</p