Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

5.2Plasminogen activator inhibitors

The proteolytic activity of uPA is physiologically regulated by plasminogen activator inhibitors (PAI), which are members of the serine proteinase inhibitor (SERPIN) family. PAI-1 and PAI-2 have been shown to interact with urokinase in a 1:1 ratio to inhibit enzyme activity and cause enzyme/inhibitor internalization and turnover.39 A role for PAI in the regulation of tumor cell invasion and motility has also been suggested, and PAI appears to be a useful prognostic marker for a number of different cancers.40

We have found significant increases in the level of PAI-1 mRNA and protein in retinas during all stages of the angiogenic response in the oxygeninduced retinopathy model.41 The functional significance of PAI-1 in retinal NV was determined by subjecting PAI-1 knockout mice to the oxygen protocol. There was about an 80% decrease in the extent of NV in these PAI- 1 knockout mice.41 The pro-angiogenic effect of PAI-1 may be explained by the fact that PAI-1 protects the stroma from excessive proteolysis during endothelial cell invasion. Excessive proteolysis during angiogenesis may prevent the coordinated assembly of endothelial cells into mature capillary tubes. A precise balance between proteolytic enzymes and their inhibitors is essential for endothelial cell migration and differentiation into functional vessels. These observations explain, at least in part, the paradoxical finding of high PAI-1 levels in advanced cancer, and thus identify PAI-1 as a potential target for anti-angiogenic therapy. It has been proposed that at low doses, PAI-1 may promote tumor growth and angiogenesis, while at higher concentrations, it may act as an anti-angiogenic agent.42 Reduction of retinal NV has been shown in an animal model by intravitreal injection of human PAI-1.43 Also, studies in choroidal NV have shown that PAI-1 can exhibit both proand anti-angiogenic effects, depending on the dose.44

The expression of both urokinase and the MMPs is modulated at the level of gene transcription by a variety of factors, including oncogenes and growth factors. Both urokinase and MMPs are secreted in a latent form and require activation. The production of the active form of these enzymes is inhibited by specific proteinase inhibitors found in the ECM. Because of these multiple levels of control, it has now become clear that these enzymes are part of a "proteolytic cascade" that functions in the regulation of cell migration and invasion, the remodeling and turnover of the ECM, and the release and activation of specific growth factors that affect cell behavior.

6.INTERACTION OF PROTEINASES WITH OTHER MOLECULES DURING OCULAR ANGIOGENESIS

6.1TNF-alpha

In addition to VEGF, other factors, including tumor necrosis factor alpha (TNFa), are expressed in the retinas of humans with proliferative eye diseases.45-47 TNFa is a 26 kDa transmembrane protein that is processed by the TNF converting enzyme, TACE, to yield a 17 kDa soluble protein.48 TNFa functions through its binding to two receptors: p55, implicated in apoptosis and NFkB (Nuclear Factor kappa B) activation, and p75, involved in lymphocyte proliferation.49,50 The cytoplasmic domain of the TNFa receptor, p55, has an 80-residue "death domain" that can regulate the apoptotic pathway.51,52 An alternative response following p55 stimulation is the activation of NFkB, which may result in a variety of cellular responses including the transcriptional regulation of select members of the MMP family of proteinases.52-54 In an animal model of retinal NV, we found increases in TNFa mRNA in the retinas on days 13 and 15.55 Isolated retinal endothelial cells did not significantly increase MMP production directly in response to a hypoxic stimulus, but required the presence of exogenous TNFa. TNFa increased the expression of MMP-3, MMP-9 and MT1-MMP in these cells. The levels of TACE and p55, proteins important in mediating the response of cells to TNFa, were increased by the angiogenic protein, VEGF, which is elevated in the retinas during NV.55 These findings support the hypothesis that growth factors such as TNFa and VEGF have a role in the regulation of extracellular proteinase expression during retinal NV.

6.2Angiopoietin

The angiopoietins are the known ligands for the Tie receptors, which are endothelial cell-specific tyrosinase kinase receptors implicated in vascular growth and development. There are four definitive members of the angiopoietin family.56 It has been hypothesized that angiopoietin 2 (Ang2) might provide a key destabilizing signal involved in initiating angiogenic remodeling. Destabilized vessels would be prone to regression in the absence of other growth factors; however, in the presence of VEGF, capillary endothelial cells are stimulated to proceed through angiogenesis.56 Increased expression of Ang2 mRNA has been shown in the retina during both normal development and NV in mice.57-59 Stimulation of cultured retinal endothelial

cells with Ang1 and Ang2 resulted in increased expression of MMP-9.59 Inhibition of the binding activity of the angiopoietins in vivo suppressed retinal NV concomitant with a reduction in the expression of MMP-9. All this evidence points toward the upregulation of MMP-9 as an early response to angiopoietin/Tek interaction, causing the destabilization of blood vessels during retinal NV.59

7.ANTI-PROTEINASE THERAPY IN TUMOR ANGIOGENESIS

Proteinase inhibitors have been used in several clinical trials in cancer because of their attractiveness as therapeutic targets. Because proteinases are expressed at the tumor site or in the surrounding stroma, the effect of these inhibitors would be localized to the tumor itself with minimal side effects.60,61 Results of several MMP inhibitors showed that although they are effective in some studies, these inhibitors work most effectively in earlystage cancer without metastasis. In fact, several clinical trials with MMP inhibitors in cancer have been terminated for lack of efficacy and the occurrence of side effects. The most severe side effect noted with MMP inhibitors is tendonitis affecting shoulder, hand, and knee joints (MMP activity is required for maintenance of adult healthy joints).60

So, what are the lessons from these cancer trials with MMP inhibitors? To design a new clinical trial with the MMP inhibitors, one needs to focus on the following questions: At what stage do the MMP inhibitors work most effectively? Which specific MMPs should be inhibited for optimal therapeutic effect? What is the role of MMPs in interaction with other proteinases, particularly uPA?

Specific MMPs play roles in specific stages of tumor progression, depending on the tissue type. For example, MMP-11 and -14 are negative prognostic indicators for small-cell lung cancer, whereas this tumor type has undetectable expression of MMP-2. So, in selecting a specific anti-MMP therapy in this cancer, one needs to use Tanomastat, which targets MMP-11 and has very little activity against MMP-2. In a transgenic mouse model of pancreatic islet cell carcinogenesis, several anti-angiogenic agents (AGM1470, angiostatin, BB-94, and endostatin) were compared for their effects at three distinct stages of cancer.62 The MMP inhibitor, BB-94, had a distinct efficacy profile in this model. It produced 49% reduction in the angiogenic islands in the prevention trial and 83% reduction in tumor burden in the intervention trial. However, it had no effect on regression of large tumors and invasive carcinoma. Thus, anti-angiogenic drugs may prove most efficacious when they are targeted to specific stages of cancer.

Since uPAR and uPA have been implicated in tumor and pathological angiogenesis, several anti-urokinase approaches have been tested in preclinical models. Anti-uPAR antibodies and the amino-terminal fragment (ATF) of uPA have been reported to inhibit tumor angiogenesis. The majority of validation studies have focused on blocking the interaction between uPA and uPAR. An oral non-cytotoxic small molecule, WX-UK1 (Wilex, Munich), an inhibitor of the uPA system, is currently in a Phase I/II clinical trial in patients with breast cancer in combination with an oral chemotherapeutic agent, Capecitabine.

8.ANTI-PROTEINASE THERAPY IN OCULAR ANGIOGENESIS

Because proteinases are attractive targets for therapy, we also tested the efficacy of several proteinase inhibitors in retinal and choroidal NV models.

8.1Reduction of Retinal NV by Inhibition of the MMPs

We used a broad-spectrum matrix metalloproteinase inhibitor, BB-94 (British Biotech Pharmaceuticals, Oxford, UK), in the retinal NV mouse model. BB-94 contains both a peptide backbone that binds it to MMPs and a hydroxamic acid group that binds it to the catalytic zinc atom they contain. Intraperitoneal (IP) injections of BB-94 have been shown to inhibit the growth of human ovarian carcinoma xenografts and murine melanoma metastasis.63,64 Upon histological examination, counts of neovascular nuclei revealed a 72% reduction in retinal NV in animals receiving a 1 mg/kg dose of BB-94 compared to control animals receiving saline as a placebo.16 The retinas of BB-94 treated animals also showed a significant decrease in the levels of active forms of MMP-2 and MMP-9, indicating that the drug reached the retinal tissues at this concentration.

8.2Reduction of Retinal NV by Inhibition of the uPA/uPAR system

For these studies, we first systemically administered a urokinase inhibitor, BB-428 (4-substituted benzo-thiophene-2-carboxamidine), which has been shown to inhibit tumor growth and invasion in models of prostate cancer and mammary adenocarcinoma.65,66 In our laboratory, we found that BB-428 inhibited retinal NV in an animal model by 58% and significantly decreased the activity of uPA in retinas of treated animals.67

A novel peptide, A6 (Angstrom Pharmaceuticals, San Diego, CA), derived from the receptor-binding region of urokinase, was also used in retinal and choroidal NV models. A6 inhibits the interaction of uPA with uPAR at the cell surface and has been shown to inhibit glioblastoma and breast cancer growth and metastasis without any direct cytotoxic effects.68,69 The anti-angiogenic activity of this peptide has been associated with a significant decrease in the density of blood vessels in these tissues. One mechanism of inhibition of new vessels may be a decrease in transforming growth factor beta activity and expression of the VEGF receptor Flk-1 as a direct or indirect result of the inhibition of the uPA-uPAR system.70 Alternatively, the uPA-uPAR interaction is required in the PAI-1 mediated recycling of uPAR and associated integrins that facilitate cell detachment from components of the ECM. Inhibition of the uPA-uPAR system by A6 might therefore be expected to disrupt this recycling process, causing increased cell-matrix adhesion and rendering the cells immobile.

The amino-terminal fragment (ATF), an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration, was used in an animal model of oxygen-induced retinopathy.71 Intravitreal injection of an adenoviral vector carrying the murine ATF reduced retinal NV by 78% in this mouse model.

We have injected the A6 peptide intraperitoneally at a dose of 5, 10, or 100 mg/kg once a day on days 12 to 16 in a model of oxygen-induced retinopathy.72 Histological analysis of mice treated with A6 peptide showed significant (63% at the highest dose) inhibition of retinal NV, and the response was dose-dependent. The reduction of NV by A6 was nearly equal to that seen in the uPAR knockout mice.

8.3Reduction of CNV by Inhibition of the MMPs

An orally administered selective MMP inhibitor, N-biphenyl sulfonylphenylalanine hydroxamic acid (BPHA), has been shown to reduce laserinduced CNV.73 In a separate experiment in a laser-induced rat CNV model, a non-peptide, small molecular weight, synthetic MMP inhibitor, AG3340 (prinomastat), was injected intravitreally to treat choroidal NV.74 Prinomastat was found to be effective when given at the time of induction of CNV in the rat model (prevention study), whereas administration of prinomastat 2 weeks after induction was not effective (regression study).

8.4Reduction of CNV by Inhibition of the uPA/uPAR system

We have found the expression of uPAR to be significantly elevated in the choroid of mice with laser-induced CNV.75 The expression of uPAR was localized specifically to the new vessels within the subretinal space associated with a disruption of Bruch’s membrane. Systemic administration of the uPA/uPAR peptide inhibitor, A6, resulted in a significant reduction of CNV (up to 94%), and the response was found to be frequencyand dosedependent (Figure 2).75 The inhibitory effects of A6 on CNV were confirmed by another group using a similar model.76 Taken together, studies on both retinal and choroidal NV demonstrate that the uPA/uPAR system is important in facilitating the development of abnormal new vessels in the retina, and thus the uPA/uPAR interaction may represent a new target for the development of anti-angiogenic therapies for ocular NV.

8.5Reduction of Retinal NV by Inhibition of the Angiopoietin/Tek system

We, along with others, reported increased expression of Ang2 in retinas during NV.57-59 To determine whether an inhibitor of the Ang2/Tek system, muTekdeltaFc (Amgen Washington, Seattle, WA), can suppress retinal NV, we injected this inhibitor intraperitoneally into experimental mice once a day (40 or 80 mg/kg) during days 12 –16. Analysis of mice treated with the Tie- 2/Tek soluble antagonist showed a significant decrease in the numbers of capillary tufts on the vitreal side of the inner limiting membrane. Quantification of neovascular nuclei showed up to 87% inhibition of retinal NV in the animal model compared to the IgG-treated control animals.59 Furthermore, this response was found to be dose-dependent.

Interestingly, RT-PCR analysis of the retinas from the Tek-treated animals showed a nearly 80% inhibition of MMP-9 expression.59 These data suggest that the upregulation of proteinases in microvascular endothelial cells by Ang2 may be an important early response during the development of retinal NV.

Figure 14-2. A6 treatment prevents NV in the laser-induced model of choroidal neovascularization (CNV). Representative images of retinal pigment epithelium–choroid whole mounts infused with fluorescein isothiocyanate–conjugated dextran 14 days after laser induction of CNV. Images are from mice treated with phosphate-buffered saline or A6 peptide at differing frequencies. The red circle roughly outlines the area of the laser burn. The fluorescence in the center of the burn area demonstrates the extent of new vessel formation under the retina. The surrounding fluorescence represents the normal choroidal vasculature. A, Mouse treated with phosphate-buffered saline twice a day for 14 days. B, Mouse treated with 100 mg/kg A6 peptide twice a day once a week. C, Mouse treated with 100 mg/kg A6 twice a day every third day. D, Mouse treated with 100 mg/kg A6 twice a day every day. E, A higher magnification image of the central region in section D. Only a few fluorescein isothiocyanate–conjugated dextran-labeled blood vessels could be seen in this area. (Reproduced from Das et al., Arch Ophthalmol 122:1844-1849, 2005, American Medical Association).

9.CONCLUSIONS

Because the upregulation and activation of proteinases and growth factors represents a final common pathway in the process of ocular NV, pharmacological intervention in these pathways may be useful as an alternative therapeutic approach to the treatment of proliferative retinopathy and exudative age-related macular degeneration.77 A clinical trial using an MMP inhibitor (AG3340, Agouron Pharmaceuticals) in patients with subfoveal CNV in age-related macular degeneration was recently terminated. We think such negative results with this MMP inhibitor do not mean that the drug is ineffective. It is possible that the drug did not reach the target tissues in effective concentrations and that the dose of the drug was insufficient. It can also be speculated that this MMP inhibitor is not effective at advanced stages of the angiogenic process and does not cause vessel regression. Based upon the pre-clinical results using A6 in our lab, two clinical trials (Phase I)

have been completed, and the drug was found to be safe and well tolerated and did not trigger any immunogenic response.78,79 The A6 will be further

tested in Phase II/III trials in patients with exudative age-related macular degeneration. As more and more novel anti-angiogenic drugs are being tested in clinical trials in patients with ocular angiogenesis, combination therapy with several anti-angiogenic drugs may be the ideal approach to completely inhibit NV, and proteinase inhibitors may play a significant role in this combination therapy.

ACKNOWLEDGMENTS

This work was supported by grant RO1 12604-07 (to A.D.) from the National Eye Institute.

REFERENCES

1.M.S. Pepper and R. Montesano, Proteolytic balance and capillary morphogenesis, Cell Differentiation & Development 32(3), 319-327, (1990).

2.P. Mignatti and D.B. Rifkin, Plasminogen activators and matrix metalloproteinases in angiogenesis, Enzyme and Protein. 49, 117-137, (1996).

3.M.S. Pepper, D. Belin, R. Montesano, L. Orci, and J.D. Vassali, Transforming growth factor beta-1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro. J. Cell Biol. 111(2), 743-755, (1990).

4.M.S. Pepper, J.D. Vassali, J.W. Wilks, L. Schweigerer, L. Orci, and R. Montesano, Modulation of microvascular endothelial cells proteolytic properties by inhibitors of angiogenesis, J. Cellular Biochem. 55(4), 419-434, (1994).

5.P.H.A. Quax, N. Pedersen, M.T. Masucci, E.J. Weening-Verhoeff, K. Dano, J.H. Verheijen, and F. Blasi, Complementation of urokinase and its receptor in extracellular matrix degradation, Cell Regulation 2, 793-803, (1991).

6.R.W. Stephens, J. Pollanen, H. Tapiovaara, K.C. Leung, P.S. Sim, E.M. Salonen, E. Ronne, N. Behrendt, K. Dano, and A. Vaheri, Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants, J. Cell Biol. 108, 1987-1995, (1989).

7.N. Manchanda and B. S. Schwartz, Single chain urokinase. Augmentation of enymatic activity upon binding to monocytes, J. Biol. Chem. 266, 14580-14584, (1991).

8.R. Blasi, Urokinase and urokinase receptor: a paracrine/autocrine system regulating cell migration and invasiveness, Bioassays 15, 105-111, (1993).

9. S.A. Rabbani, J. Desjardins, A.W. Bell, D. Banville, A. Mazar, J. Henkin, and

D.Goltzman, An amino-terminal fragment of urokinase isolated from a prostate cancer cell line is mitogenic for osteoblast-like cells Biochem. Biophys. Res. Commun. 173, 1058-1064, (1990).

10.S. A. Rabbani and A. P. Mazar, The role of plasminogen activation system in angiogenesis and metastasis, Surg. Oncol. Clin. N. Amer. 10, 393-415, (2001).

11.Y. Wei, D. A. Waltz, N. Rao, R. J. Drummond, S. Rosenberg, and H. A. Chapman, Identification of the urokinase receptor as an adhesion receptor for vitronectin, J. Biol. Chem. 269, 32380-32388, (1994).

12.F. Blasi and P. Carmeliet, uPA: a versatile signaling orchestrator, Nat. Rev. Mol. Cell. Biol. 3, 931-943, (2002).

13.W. G. Stetler-Stevenson, The role of matrix metalloproteinases in tumor invasion, metastasis, and angiogenesis, Surg. Oncol. Clin. N. Amer. 10, 383-392, (2001).

14.M. S. Pepper, Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis, Arterioscler. Thromb. Vasc. Biol. 21, 1104-1117, (2001).

15.M. Egeblad and Z. Werb, New functions for the matrix metalloproteinases in cancer progression, Nat. Rev. Cancer 2, 161-174, (2002).

16.A. Das, A. McLamore, W. M. Song, and P. G. McGuire, Retinal neovascularization is suppressed with a matrix metalloproteinase inhibitor. Arch. Ophthalmol. 117, 498-503, (1999).

17.M. Hangai, N. Kitaya, J. Xu, C. K. Chan, J. J. Kim, Z. Werb, S. J. Ryan, and P. C. Brooks, Matrix metalloproteinase-9-dependent exposure of a cryptic migratory control site in collagen is required before retinal angiogenesis, Am. J. Pathol. 161, 1429-1437, (2002).

18.P. G. McGuire, T. R. Jones, N. Talarico, E. Warren, and A. Das, The Urokinase/ Urokinase Receptor System in Retinal Neovascularization: Inhibition by Å6 Suggests A New Therapeutic Target. Invest. Ophthalmol. Vis. Sci. 44, 2736-2742, (2003).

19.A. Das, P. G. McGuire, C. Eriqat, R. R. Ober, E. DeJuan, G. A. Williams, A. McLamore,

J.Biswas, and D. W. Johnson, Human diabetic neovascular membranes contain high levels of urokinase and metalloproteinase enzymes, Invest. Ophthalmol. Vis. Sci. 40, 809-813, (1999).

20.V. Lambert, C. Munaut, P. Carmeliet, R. D. Gerard, P. Declerck, A. Gils, C. Claes,

J.M. Foidart, A. Noel, and J. M. Rakic, Dose-dependent modulation of choroidal neovascularization by plasminogen activator inhibitor type 1: Implications for clinical trials, Invest. Ophthalmol. Vis. Sci. 44, 2791-2797, (2003).

21.A. Das, N. Boyd, T. R. Jones, N. Talarico, and P. G. McGuire, Inhibition of Choroidal Neovascularization by a Peptide Inhibitor of the Urokinase Plasminogen Activator and Receptor System in a Mouse Model, Arch. Ophthalmol. 122, 1844-1849, (2004).

22.S. Giebel, G. Menucucci, P. McGuire, and A. Das, Matrix Metalloproteinases in Early Diabetic Retinopathy and Their Role in Alteration of the Blood-Retinal Barrier. Lab. Invest. 85, 597-607, (2005).

23.A. El-Remessy, M. A. Behzadian, G. Abou-Mohamed, T. Franklin, R. W. Caldwell, and R. B. Caldwell, Experimental diabetes causes breakdown of the blood-retinal barrier by a mechanism involving tyrosine nitration and increases in expression of vascular endothelial growth factor and urokinase plasminogen activator receptor. Am. J. Pathol. 162, 1995-2004 (2003).

24.D. A. Antonetti, A. J. Barber, S. Khin, E. Lieth, J. M. Tarbell, and T. W. Gardner, Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content, Diabetes 47, 1953-1959, (1998).

25.D. A. Antonetti, A. J. Barber, L. A. Hollinger, E. B. Wolpert, and T. W. Gardner, Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occuden, 1. J. Biol. Chem. 274, 23463-23467, (1999).

26.Herron, M. J. Banda, E. J. Clark, J. Gavrilovic, and Z. Werb, Secretion of MMPs by stimulated capillary endothelial cells: expression of collagenases and stromelysin activities is regulated by endogenous inhibitors, J. Biol. Chem. 261, 2814-2818, (1986).

27.E. Baramova and J. M. Foidart, Matrix metalloproteinase family, Cell Biol. Int. 19, 239-242, (1995).

28.M. Moses, The regulation of neovascularization by MMPs and their inhibitors. Stem Cells 15, 180-189, (1996).

29.M. A. Moses and R. A. Langer, A metalloproteinase inhibitor as an inhibitor of neovascularization, J. Cell. Biochem. 47, 230-235, (1991).

30.P. D. Brown, D. E. Kleine, E. J. Unsworth, and W. G. Stetler-Stevenson, Cellular activation of the 72 kD type IV procollagenase:TIMP-2 complex, Kidney Int. 43, 163-168, (1993).

31.D. Kleiner, A. Tuuttila, K. Tryggvason, and W. Stetler-Stevenson, Stability analysis of latent and active 72 kD type IV collagenase:the role of tissue inhibitor of metalloproteinase-2 (TIMP-2), Biochemistry 32, 1583-1587, (1993).

32.R. N. Farris, S. S. Apte, B. R. Olsen, K. Iwata, and A. H. Milam, Tissue inhibitor of metalloproteinase-3 is a component of Bruch’s membrane of the eye, Am. J. Pathol. 150, 323-328, (1997).

33.N. Della, P. Campochiaro, and D. Zack, Localization of TIMP-3 mRNA to the retinal pigment epithelium, Invest. Ophthalmol. Vis. Sci. 37, 1921-1924, (1996).

34.A. Ruiz, P. Brett, and D. Bok, TIMP-3 is expressed in the human retinal pigment epithelium, Biochem. Biophys. Res. Commun. 226, 467-474, (1996).

35.B. H. F. Weber, G. Vogt, R. C. Pruett, H. Stohr, and U. Felbor, Mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) in patients with Sorsby’s fundus dystrophy, Nat. Genet. 8, 352-356, (1994).

36.R. N. Farris, S. S. Apte, P. J. Luthert, A. Bird, and A. H. Milam, Accumulation of tissue inhibitor metalloproteinase-3 in human eyes with Sorsby’s fundus dystrophy or retinitis pigmentosa, Br. J. Ophthalmol. 82, 1329-1334, (1998).

37.M. Kamei and J. G. Hollyfield, TIMP-3 in Bruch’s membrane:changes during aging and age-related macular degeneration, Invest. Ophthalmol. Vis. Sci. 40, 2367-2375, (1999).

38.S. Majka, P. G. McGuire, S. Colombo, and A. Das, The balance between proteinases and inhibitors in a murine model of proliferative retinopathy. Invest. Ophthalmol. Vis. Sci. 42, 210-215, (2001).