In vivo evidence for an endothelium-dependent mechanism in radiation-induced normal tissue injury

Abstract

The pathophysiological mechanism involved in side effects of radiation therapy, and especially the role of the endothelium remains unclear. Previous results showed that plasminogen activator inhibitor-type 1 (PAI-1) contributes to radiation-induced intestinal injury and suggested that this role could be driven by an endothelium-dependent mechanism. We investigated whether endothelial-specific PAI-1 deletion could affect radiation-induced intestinal injury. We created a mouse model with a specific deletion of PAI-1 in the endothelium (PAI-1KO(endo)) by a Cre-LoxP system. In a model of radiation enteropathy, survival and intestinal radiation injury were followed as well as intestinal gene transcriptional profile and inflammatory cells intestinal infiltration. Irradiated PAI-1KO(endo) mice exhibited increased survival, reduced acute enteritis severity and attenuated late fibrosis compared with irradiated PAI-1(flx/flx) mice. Double E-cadherin/TUNEL labeling confirmed a reduced epithelial cell apoptosis in irradiated PAI-1KO(endo). High-throughput gene expression combined with bioinformatic analyses revealed a putative involvement of macrophages. We observed a decrease in CD68(+)cells in irradiated intestinal tissues from PAI-1KO(endo) mice as well as modifications associated with M1/M2 polarization. This work shows that PAI-1 plays a role in radiation-induced intestinal injury by an endothelium-dependent mechanism and demonstrates in vivo that the endothelium is directly involved in the progression of radiation-induced enteritis.

Figure 1. Generation of endothelium-specific PAI-1 knockout mice.

Molecular targeting strategy. Primers P1 to P4 used for PCR analysis are indicated on alleles. P1-P2 and P1-P3 products are used for mice genotyping, while P2-P4 products are used for neo-cassette excision checking. ex = exon; DTA = diphtheria toxin A fragment gene; neo = neomycin cassette; FLP = flip-flop recombinase; FRT = Flp recognition target; loxP = locus of X-over P1; Cre = cyclic recombinase; ATG = start codon; STP = stop codon.

Figure 2. PAI-1 endothelial deletion limits radiation-induced up-regulation of intestinal PAI-1 expression and protects mice from death in a radiation-induced enteritis model.

(a) Relative PAI-1 mRNA level was measured by RT-qPCR in intestinal tissue in PAI-1^flx/flx sham-IR, and in irradiated PAI-1^flx/flx and PAI-1KO^endo mice. Results are means ± SEM with *P < 0.05,**P < 0.01 and ***P < 0.001 with n = 8 to 12 mice per group. (b) Kaplan-Meier analyses representing the percent survival of irradiated PAI-1^(flx/flx) mice and PAI-1KO^endo mice. The log rank test was used for statistical analyses with NS, non-significant, *P < 0.05 and ***P < 0.001.

Figure 3. Endothelial-specific PAI-1 deletion limits acute radiation enteritis.

(a) Representative microscopic alterations obtained in PAI-1^flx/flx and PAI-1KO^endo 3 days after irradiation. Slides were stained with hematoxylin-eosin-saffron (upper panels) or with antibody against E-cadherin (red) and counterstained with DAPI (blue) (lower panels). Scale bar = 100 μm. (b) The number of crypts as well as the severity of cryptic damage were evaluated for each group. The number of crypts is expressed as a percentage of sham-IR mice. ***P < 0.001 versus PAI-1^flx/flx sham-IRmice; ^#P < 0.01 versus PAI-1^flx/flx/19 Gy mice (8 to 12 mice per group). For each group, crypts are categorized according to severity of their damage. Lesions range from grade 0 (no lesion) to 3 (phantom crypt). Results are expressed as a percentage of total crypts. (c) Representative microscopic alterations obtained in PAI-1^flx/flx and PAI-1KO^endo 7 days after irradiation.(d) Parameters of mucosal regeneration were evaluated. Results are expressed as a percentage of mice showing these parameters with 8 to 12 mice per group. (e) Evaluation of the severity of muscularis propria inflammation. Scoring ranges from 0 (no lesion) to 4 (loss of muscularis propria). *P < 0.01.

Figure 4. Endothelial-specific PAI-1 deletion reduces acute radiation-induced epithelial cell death.

(a) Gene expression profiles (5 hours after irradiation) with significant differences between sham-IR and irradiated mice were visualized by a heat map. (b) Venn diagram of genes with a significant mRNA level modification in irradiated PAI-1^flx/flx and PAI-1KO^endo mice compared with the sham-IR group. (c) Representative microscopic alterations obtained in PAI-1^flx/flx mice and PAI-1KO^endo mice, irradiated or not. Slides were double-stained with antibody against E-cadherin (red) and TUNEL labeling (green), then counterstained with DAPI (blue). Scale bar = 100 μm. (d) The number of apoptotic cells in crypts was evaluated for each group (n = 6 mice per group). Results are expressed as number of epithelial apoptotic cells per crypt. ***P < 0.001 versus PAI-1^flx/flx sham mice; ^#P < 0.01 versus PAI-1^flx/flx 19 Gy.

Figure 5. Constitutive and inducible endothelial-specific PAI-1 deletion limits fibrosis following a single high-dose radiation exposure.

(a) Representative microscopic alterations obtained in PAI-1^flx/flx mice and PAI-1KO^endo mice 6 weeks after irradiation. Slides were stained with hematoxylin-eosin-saffron (upper panels) or Sirius red (lower panels). Scale bar = 100 μm n = 5 for PAI-1^flx/flxsham-IR mice; n = 8 for other groups. Fibrosis score in constitutive (b) or inducible (c) PAI-1KO^endo mice (named PAI-1KO^endo(i)). Scores ranged from 0 (no damage) to 4 (severe fibrosis). All sham-IR mice displayed a score of 0 (not shown). For experiments with inducible mice, the 3 groups were treated in the same conditions with tamoxifen. n = 5 for PAI-1^flx/flx sham-IR mice (scores of 0 are not shown) and n = 8 to 11 for the other groups. *P < 0.05.

Figure 6. Endothelial-specific PAI-1 deletion impacts the molecular profile associated with immune-related genes in irradiated intestinal tissue.

Gene expression profiles 3 days (a) and 7 days (b) after irradiation showing significant differences between sham-IR and irradiated mice are visualized in the heat map. (c,d) Corresponding Venn diagrams of genes with a significant change in mRNA level in irradiated PAI-1^flx/flx mice and PAI-1KO^endo mice compared with the sham-IR group.

Figure 7. Conditional endothelium-specific PAI-1 deletion limits macrophage infiltration and influences macrophage M1/M2 polarization.

(a) Representative labeling of macrophages in intestinal tissue 7 days after irradiation. Slides were stained with antibodies against CD68 (blue) and counterstained with nuclear fast red (pink). Scale bar = 100 μm. (b) Macrophage scoring. Scores ranged from 0 (sham-IR) to 4 (maximum macrophage count). n = 6 for sham-IRPAI-1^flx/flxmice, n = 8 for PAI-1^flx/flx 19 Gy mice, and n = 6 for PAI-1KO^endo19 Gy mice. *P < 0.01; (c) Representative double labeling of M1 macrophages in intestinal tissue 1 week after irradiation. Slides were stained with antibodies against CD68 (red) and iNOS (green) and counterstained with DAPI. (d) Quantification of M1 macrophages (yellow merging signal) in sham-IR PAI-1^flx/flxmice, PAI-1^flx/flx 19 Gy mice and PAI-1KO^endo19 Gy mice at 3, 7 and 42 days after irradiation. *P < 0.05 ND: not detected in sham-IR mice. (e) Representative double labeling of M2 macrophages in intestinal tissue 7 days after irradiation. Slides were stained with antibodies against CD68 (red) and CD206 (green) and counterstained with DAPI. (f) Quantification of M2 macrophages (yellow merging signal) in sham-IR PAI-1^flx/flx mice, PAI-1^flx/flx19 Gy mice and PAI-1KO^endo19 Gy mice at 3, 7 and 42 days after irradiation. For all experiments, n = 6 for sham-IRPAI-1^flx/flxmice, n = 8 for PAI-1^flx/flx19 Gy mice, and n = 6 for PAI-1KO^endo19 Gy mice. *P < 0.05.