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, 117 (6), 1514-26

Strain-dependent Embryonic Lethality and Exaggerated Vascular Remodeling in Heparin Cofactor II-deficient Mice

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Strain-dependent Embryonic Lethality and Exaggerated Vascular Remodeling in Heparin Cofactor II-deficient Mice

Ken-ichi Aihara et al. J Clin Invest.

Abstract

Heparin cofactor II (HCII) specifically inhibits thrombin action at sites of injured arterial wall, and patients with HCII deficiency exhibit advanced atherosclerosis. However, the in vivo effects and the molecular mechanism underlying the action of HCII during vascular remodeling remain elusive. To clarify the role of HCII in vascular remodeling, we generated HCII-deficient mice by gene targeting. In contrast to a previous report, HCII(-/-) mice were embryonically lethal. In HCII(+/-) mice, prominent intimal hyperplasia with increased cellular proliferation was observed after tube cuff and wire vascular injury. The number of protease-activated receptor-1-positive (PAR-1-positive) cells was increased in the thickened vascular wall of HCII(+/-) mice, suggesting enhanced thrombin action in this region. Cuff injury also increased the expression levels of inflammatory cytokines and chemokines in the vascular wall of HCII(+/-) mice. The intimal hyperplasia in HCII(+/-) mice with vascular injury was abrogated by human HCII supplementation. Furthermore, HCII deficiency caused acceleration of aortic plaque formation with increased PAR-1 expression and oxidative stress in apoE-KO mice. These results demonstrate that HCII protects against thrombin-induced remodeling of an injured vascular wall by inhibiting thrombin action and suggest that HCII is potentially therapeutic against atherosclerosis without causing coagulatory disturbance.

Figures

Figure 1
Figure 1. Targeted disruption of murine HCII gene, genotyping, and karyotypes of HCII-mutant mice.
(A) Genomic locus, targeting vector, and predicted targeting locus are illustrated. External (probe A) and internal (probe B) probes were used for Southern blot analysis. Two sets of PCR primers for detecting the 480-bp WT allele and 740-bp mutant allele were employed for genotyping. (B) Southern blot analysis of murine genomic DNA. A 5-kbp HindIII fragment denotes the homologous recombinant allele in probe A as an external probe, and a single 7-kbp HindIII fragment denotes the homologous recombinant allele without random integration in probe B as an internal probe. (C) Genotyping PCR. The mutant allele yielded a 740-bp band, and the WT allele yielded a 480-bp band. (D) Karyotype analysis of HCII+/– male and female mice. Chromosomes of splenic lymphocytes in both male and female HCII mutant mice showed normal karyotypes.
Figure 2
Figure 2. Noninterference of HCII targeting vector introduction into murine genome.
(A) FISH analysis using a phosphoglycerate kinase–neomycin cassette probe and genomic map around the HCII genome. Single green spots in male and female HCII+/– murine chromosomes indicate no random integration and adequate homologous recombination of the HCII targeting vector. Original magnification, ×1,000; ×4,000 (insets). (B) Real-time PCR analysis for mRNA quantification of genes located around the HCII genome in HCII+/+ (white bars) and HCII+/– mice (black bars). Values were normalized by arbitrarily setting the measurement for HCII+/+ mice to 1.0. Ube2l3, ubiquitin-conjugating enzyme E2L3; Pik4ca, phosphatidylinositol 4-kinase, catalytic, alpha polypeptide; Snap29, synaptosomal-associated protein.
Figure 3
Figure 3. Analysis of HCII gene transcripts, protein, and plasma activity.
(A) Conventional RT-PCR analysis of full-length HCII gene. (B) Western blot analysis of hepatic HCII protein in HCII+/+ and HCII–/– mice. (C) Northern blot analysis of hepatic HCII mRNA in HCII+/+ and HCII+/– mice. (D) Plasma HCII activity in HCII+/+ (white bar) and HCII+/– mice (black bar). Values are expressed as mean ± SEM. **P < 0.01. n = 12 in each group for plasma HCII activity.
Figure 4
Figure 4. Effect of HCII deficiency on hemostatic examinations.
(A) Plasma fibrinogen concentration, prothrombin time, and AT activity in HCII+/+ (white bars) and HCII+/– mice (black bars). Values are expressed as mean ± SEM. n = 6–12 in each group. (B) The left and middle panels show representative results of platelet aggregation analyzed by the screen filtration pressure method in HCII+/+ and HCII+/– mice. The platelet aggregation pressure of each reaction tube was determined as the pressure rate (percentage). For screen filtration pressure aggregometer study, the pressure rate was standardized using a grading curve produced by plotting 3 concentrations of ADP on the x axis and pressure rate (percentage) on the y axis. The concentration of ADP causing a 50% increase in pressure rate was calculated and applied as the platelet aggregatory threshold index (PATI) in HCII+/+ (white bar) and HCII+/– mice (black bar). n = 6 in each group. Values are expressed as mean ± SEM. *P < 0.05.
Figure 5
Figure 5. Histopathology of femoral arteries with and without cuff injury in HCII+/+ and HCII+/– mice.
(A) Histological features of uninjured and cuff-injured femoral arteries. Cross sections of uninjured and cuff-injured arteries were obtained on day 28 of cuff injury and stained with Elastica–van Gieson. (B) Quantitative analysis of arterial intimal area, medial area, adventitial area, intima/media ratio, and adventitia/media ratio with and without cuff injury in HCII+/+ (white bars) and HCII+/– mice (black bars). Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01. n = 12 in each group.
Figure 6
Figure 6. Immunostaining of proliferative vascular mesenchymal cells with PCNA and BrdU at the vascular wall with and without cuff injury in HCII+/+ and HCII+/– mice.
(A) PCNA staining of the vascular wall with and without cuff injury in HCII+/+ and HCII+/– mice. (B) Quantitative analysis of PCNA-positive cells in intima and media with and without cuff injury in HCII+/+ (white bars) and HCII+/– mice (black bars). Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01. n = 8 in each group. (C) BrdU staining of the vascular wall with and without cuff injury in HCII+/+ and HCII+/– mice. (D) Quantitative analysis of BrdU-positive cells in intima and media with and without cuff injury in HCII+/+ (white bars) and HCII+/– mice (black bars) Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01. n = 8 in each group.
Figure 7
Figure 7. Expression of PAR-1, inflammatory cytokines and chemokines, and vascular remodeling–related transcription factors with and without cuff injury in HCII+/+ and HCII+/– mice.
(A) Immunohistochemical staining of PAR-1 with and without cuff injury in HCII+/+ and HCII+/– mice. (B) Gene expression of vascular remodeling factors at the vascular wall with and without cuff injury in HCII+/+ (white bars) and HCII+/– mice (black bars). Real-time PCR analyses of Il1b, Il6, Mcp1, Egr1, and Klf5 were performed in HCII+/+ and HCII+/– mice. Values were normalized by arbitrarily setting the measurement in HCII+/+ mice without cuff injury to 1.0. Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01. n = 10 in each group.
Figure 8
Figure 8. Effects of human purified HCII supplementation on cuff-injured vascular remodeling in HCII+/– mice.
(A) Time course of plasma HCII activity in HCII+/– mice after an intraperitoneal injection of human purified HCII protein (h-HCII) (open circles) or a vehicle alone (filled circles). *P < 0.05. n = 4 in each group. (B) Representative histological findings of cuff-injured femoral arteries. Micrographs of cross sections of cuff-injured arteries stained with Elastica–van Gieson on day 28 in HCII+/+ mice treated with vehicle and in HCII+/– mice treated with vehicle alone or human purified HCII protein are shown. (C) Quantitative comparison of arterial intimal area, medial area, adventitial area, intima/media ratio, and adventitia/media ratio in cuff-injured HCII+/+ mice treated with vehicle (white bars) and HCII+/– mice treated with vehicle (black bars) or human purified HCII protein (gray bars). Values are expressed as mean ± SEM. *P < 0.05; **P < 0.01. n = 10 in each group.
Figure 9
Figure 9. Effects of HCII deficiency and of human purified HCII supplementation on wire-injured vascular remodeling.
(A) Representative histological findings for wire-injured femoral arteries. Micrographs of cross sections of wire-injured arteries stained with Elastica–van Gieson on day 28 in HCII+/+ mice treated with vehicle and in HCII+/– mice treated with vehicle alone or human purified HCII protein are shown. (B) Quantitative comparison of arterial intimal area, medial area, adventitial area, intima/media ratio, and adventitia/media ratio in wire-injured HCII+/+ mice treated with vehicle (white bars) and HCII+/– mice treated with vehicle (black bars) or human purified HCII protein (gray bars). Values are expressed as mean ± SEM. *P < 0.05. n = 8–10 in each group.
Figure 10
Figure 10. Quantification and characterization of atherosclerotic lesions in HCII+/+apoE–/– and HCII+/–apoE–/– mice.
(A) The left panel shows representative histological findings in Masson trichrome–stained serial sections of the aortic root in HCII+/+apoE–/– and HCII+/–apoE–/– mice. The right panel shows mean values of the atherosclerotic plaque area in HCII+/+apoE–/– (white bar) and HCII+/–apoE–/– mice (black bar). Values are expressed as mean ± SEM. *P < 0.05. n = 12 in each group. (B) Representative histological findings in oil red O– (upper panels) and PAR-1–stained (lower panels) serial sections of the aortic root in HCII+/+apoE–/– and HCII+/–apoE–/– mice.
Figure 11
Figure 11. Effect of HCII deficiency on oxidative stress in apoE–/– mice.
(A) Representative findings in serial sections of the aortic root stained with DHE in HCII+/+apoE–/– and HCII+/–apoE–/– mice. (B) Mean values of urinary excretion of 8OHdG in HCII+/+apoE–/– (white bar) and HCII+/–apoE–/– mice (black bar). Values are expressed as mean ± SEM. **P < 0.01. n = 6 in each group.

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