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. 2004 Jan;113(1):38-48.
doi: 10.1172/JCI19684.

Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo

Qi Ling  1 , Andrew T Jacovina, Arunkumar Deora, Maria Febbraio, Ronit Simantov, Roy L Silverstein, Barbara Hempstead, Willie H Mark, Katherine A Hajjar
Affiliations

Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo

Qi Ling et al. J Clin Invest. 2004 Jan.

Abstract

A central tenet of fibrinolysis is that tissue plasminogen activator-dependent (t-PA- dependent) conversion of plasminogen to active plasmin requires the presence of the cofactor/substrate fibrin. However, previous in vitro studies have suggested that the endothelial cell surface protein annexin II can stimulate t-PA-mediated plasminogen activation in the complete absence of fibrin. Here, homozygous annexin II-null mice displayed deposition of fibrin in the microvasculature and incomplete clearance of injury-induced arterial thrombi. While these animals demonstrated normal lysis of a fibrin-containing plasma clot, t-PA-dependent plasmin generation at the endothelial cell surface was markedly deficient. Directed migration of annexin II-null endothelial cells through fibrin and collagen lattices in vitro was also reduced, and an annexin II peptide mimicking sequences necessary for t-PA binding blocked endothelial cell invasion of Matrigel implants in wild-type mice. In addition, annexin II-deficient mice displayed markedly diminished neovascularization of fibroblast growth factor-stimulated cornea and of oxygen-primed neonatal retina. Capillary sprouting from annexin II-deficient aortic ring explants was markedly reduced in association with severe impairment of activation of metalloproteinase-9 and -13. These data establish annexin II as a regulator of cell surface plasmin generation and reveal that impaired endothelial cell fibrinolytic activity constitutes a barrier to effective neoangiogenesis.

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Figures

Figure 1
Figure 1
Generation of annexin II–null mice. (a) Genomic organization of the wild-type and targeted alleles after homologous recombination. The location of the probe used in Southern blotting is shown (arrow), as well as the sizes of EcoRI fragments identified for wild-type and mutant alleles (double headed arrows). Numerals 1–5 refer to exons 1–5. E, EcoRI; H, HindIII; S, SpelI; B, BlinI. (b) Southern blot analysis of DNA obtained from tail tips. Fragments corresponding to wild-type (18.8 kb) and targeted (16.4 kb) alleles are shown. (c) Northern blot analysis of total RNA from lung and heart. RNA loading was monitored by ethidium bromide staining of RNA. (d and e) Western blots utilizing monoclonal (d) and polyclonal (e) IgG directed against human annexin II. Protein expression for all three genotypes is shown. Con, control.
Figure 2
Figure 2
Fibrin deposition. (a) Western blot analysis of five tissues showing mouse fibrin standards (Stds) in lanes 1–3 (2, 5, and 10 ng for lung, spleen, and small intestine [Sm. int.], and 1, 2, and 5 ng for liver and kidney). Samples from four separate annexin II–null and wild-type mice are shown in lanes 4–7 and 8–11, respectively. (b) Quantitative analysis of combined fibrin deposition data (mean ± SE, n = 4 experiments). (c) Immunohistochemical analysis of wild-type and annexin II–null lung, myocardium, and kidney showing fibrin deposition in renal glomeruli (red arrowheads), myocardial capillaries (black arrowheads), and alveolar capillaries (red arrowheads). Scale bars indicate 0.05 mm (kidney) and 0.025 mm (heart and lung).
Figure 3
Figure 3
Fibrinolytic function. (a) Plasma clot lysis profiles in the presence (+) and absence (–) of t-PA in wild-type and annexin II–null mice. A, absorbance. (b) Carotid blood flow before and after a 3-minute application of 10% FeCl3 (*) in wild-type and annexin II–null animals. (c) Maximal arterial occlusion, as % of initial flow rate, following application of FeCl3 in wild-type and annexin II–null animals (mean ± SE, n = 12). (d) Maximal recovery of blood flow over 30 minutes, as % of initial flow rate, in wild-type and annexin II–null animals (mean ± SE, n = 11).
Figure 4
Figure 4
Endothelial cell function. (a) VEGF-A-directed migration of wild-type (white bars) and annexin II–deficient (black bars) microvascular endothelial cells across collagen I and fibrin barriers. Shown are mean ± SE, n = 5 experiments. (b) Thoracic aortic rings embedded in type I collagen gels and incubated in serum-containing medium for 6 days. Shown are phase-contrast images of branching vessel-like structures in wild-type and annexin II–deficient explants. Scale bar, 0.25 mm. (c) t-PA-dependent plasmin generation in the presence of wild-type (filled circles) versus annexin II–null (open circles) microvascular endothelial cells expressed as relative fluorescence units (RFU). (d) MMP-9 activity in medium conditioned by aortic ring explants from wild-type (white bars) and annexin II–null (black bars) mice in the absence (–PLG) and presence (+PLG) of added plasminogen (2 μg/ml). Shown are mean ± SE, n = 6 experiments. (e) Media from annexin II–deficient (KO) and wild-type aortic ring explants obtained after culture, analyzed by Western blot using monoclonal anti–MMP-13 IgG and chemiluminescence. The putative active MMP-13 fragment migrates at about 48 kDa and proMMP-13, at about 60 kDa. (f) Densitometric analysis of MMP-13 Western blots of medium from cultures of wild-type (white bars) and annexin II–deficient (black bars) aortic rings. Shown are mean ± SE, n = 5 experiments.
Figure 5
Figure 5
Neovascularization of Matrigel plugs. (a) Annexin II immunostaining. Matrigel plugs containing VEGF-A (2 μg/ml) were implanted in wild-type mice, harvested on day 12, and stained with no primary antibody (Con), anti-annexin II (A II) IgG, or anti-annexin IV (A IV) IgG. Scale bar indicates 0.05 mm. (b) Neovascularization in annexin II–deficient mice. Matrigel plugs containing VEGF-A (100 ng/ml) or bFGF (100 ng/ml) were implanted in wild-type and annexin II–deficient mice, harvested on day 12, fixed, sectioned, and stained with H&E. Scale bar indicates 0.05 mm. (c) Effect of annexin II N-terminal peptide. Matrigel plugs contained either sense peptide (STVHEILCKLSL; 250 μM), mimicking sequences required for t-PA binding to annexin II, or a scrambled control peptide (SLTSVLHKECLI; 250 μM) were analyzed on day 12 by enumeration of cells in sections stained with H&E (10 random fields per section, 10 sections per sample, four mice per condition). Shown are mean cells per 200× field (SE; n = 4 mice). (d) Histological analysis of Matrigel plugs containing scrambled or sense tail peptide (H&E stain). Scale bar indicates 0.05 mm.
Figure 6
Figure 6
Corneal neoangiogenesis. (a) Neovascular response. Corneas of wild-type and annexin II–null littermates were implanted with pellets containing 0–50 ng bFGF and were photographed on day 5. Original magnification, ×75. (b) Angiogenesis index. The corneal response was quantified for eight eyes at each of five doses of bFGF. (c) Corneal vessel histology. Immunohistochemical analysis of frozen corneal sections stained with anti-fibrin(ogen)/alkaline phosphatase (blue) and anti-PECAM/peroxidase (brown) show partial-thickness fibrin deposition (arrowheads) and vessel-associated endothelial cells (red arrow) in wild-type versus full-thickness fibrin (arrowheads) and isolated endothelial cells (red arrow) in annexin II–deficient corneas. Scale bar, 0.1 mm.
Figure 7
Figure 7
Oxygen-induced retinopathy. (a) UV fluorescence images of en face preparations of FITC-dextran–perfused retinas of wild-type (+/+) and annexin II–null (–/–) mice treated with oxygen (O2) or maintained in room air (RA). Original magnification, ×40. (b) H&E–stained sections of retinas froot annexin II–deficient mice exposed to high oxygen. Original magnification, ×400. Arrowheads indicate dilated intraretinal blood vessels. (c) Retinal neovascular tufts in wild-type mice induced by high oxygen exposure were stained with anti–annexin II (A II) IgG and isotype-matched anti–annexin IV (A IV) IgG, and counterstained with hematoxylin (original magnification, ×1,000). (d) Quantification of vascular nuclei (left panel) and vascular tufts (right panel) located beyond the ILM in retinas of wild-type and annexin II–null newborn mice treated with high oxygen or maintained in room air, at day 17. Shown are mean ± SE (n = 4 experiments).
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