KR20010022887A - Stimulation, modulation and/or inhibition of endothelial proteolytic activity and/or angiogenic activity - Google Patents

Abstract

Vascular endothelial growth factor-B (VEGF-B) and vascular endothelial growth factor-C (VEGF-C) are angiogenic polypeptides. It has been known that VEGF-B and -C are angiogenic in the laboratory, especially in combination with bFGF. VEGF-C also increases plasminogen activator (PA) activity in bovine endothelial cell lines, which is accompanied by a simultaneous increase in PA inhibitor-1. The addition of alpha-2-antiplasmin to bovine endothelial cells co-treated with bFGF and VEGF-C partially inhibits collagen invasion.

Description

Stimulation, regulation and / or inhibition of endothelial proteolytic and / or angiogenic activity {STIMULATION, MODULATION AND / OR INHIBITION OF ENDOTHELIAL PROTEOLYTIC ACTIVITY AND / OR ANGIOGENIC ACTIVITY}

Angiogenesis is renal capillary formation by processes that develop from already existing blood vessels, and occurs not only during development but in many physiological and pathological environments. Thus, angiogenesis is essential for tissue growth, wound healing and female reproductive function, and is also a component of pathological processes such as tumor growth, hemangioma formation and neovascularization of the eye [Folkman, New Engl. J. Med., 333: 1757-1763 (1995); Pepper, Arterioscler. Thromb. Vasc. Biol., 17: 605-619 (1997). A similar but much less studied process also occurs in the lymphatic system and is often referred to as lymphangiogenesis.

Angiogenesis begins with localized destruction of the basement membrane of the parent vessels, which then leads to migration and growth of endothelial cells into the surrounding extracellular epilepsy and results in the formation of capillary development buds. The lumen is then formed and constitutes an essential element in the formation of functional germinal shoots. Developmental shoot maturation is completed after reconstitution of the basement membrane. During this series of events, at least three endothelial cell functions are altered: 1) regulation of interaction with extracellular epilepsy, alteration of cell-stromal contact and interstitial-degrading proteolytic enzymes (plasminogen activators (PAs) ) And interstitial metalloproteinases); 2) the initial increase and subsequent decrease in migration (migration), which causes the cells to move towards angiogenic stimulation and stop once they arrive at their designated location; 3) Increase in proliferation, which provides renal cells to grow and prolong blood vessels and then return to dormant once the vessels are formed. Together, these cell functions perform capillary morphogenesis, ie the formation of three-dimensional open or open tube-like structures. Many newly formed capillaries then undergo vascular wall maturation processes (ie, the formation of smooth muscle cell-rich media and outer membranes), while others undergo degeneration (ie, absence of blood flow) [Pepper et al., Enzyme Protein, 49: 138 -162 (1996); Risau, Nature 386: 671-674 (1997).

Except for angiogenesis, which occurs in response to tissue damage or in the female reproductive organs, it is generally mentioned that endothelial cell turnover is very low in healthy adult organisms. The persistence of the endothelium is thought to be due to the presence of endogenous negative regulators, since positive regulators are often detected in adult tissues that are clearly devoid of angiogenesis. The reverse is also true: positive and negative regulators often coexist in tissues with increased endothelial cell replacement. This leads to the concept of "switch of angiogenesis", in which the endothelial activity is determined by the balance between positive and negative regulators. Positive regulators prevail in activated (angiogenic) endothelium, while dominant negative regulators perform and sustain endothelial pacing (Hanahan et al., Cell, 86: 353-364 (1996)). Initially used in the context of tumor progression to describe delivery from the anterior vasculature to the vasculature, the concept of a "switch" can be applied not only to pathological angiogenesis but also to developmental and physiological contexts. While it remains limited argument in vivo, the hypothesis under discussion is that "switches" are related to the induction of positive regulators and / or the loss of negative regulators.

Many polypeptide growth factors or cytokines have been demonstrated to be angiogenic in vivo [Klagsbrun et al., Ann. Rev. Physiol., 53: 217-39 (1991); Leek et al., J. Leukocyte Biol., 56: 423-435 (1994); Pepper et al., Curr. Topics Microbiol. Immunol., 213 / II: 31-67 (1996). These also include vasculature endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), known as permeability factor or vasculotropin of the vasculature. However, while the role of VEGF in the development of embryonic vasculature and tumor angiogenesis is clearly established [Carmeliet et al., Nature, 380: 435-39 (1996); Ferrara et al., Nature, 380: 439-442 (1996); Dvorak et al., Amer. J. Pathol., 146: 1029-1039 (1995); Ferrara et al., Endocrine Rev., 18: 4-25 (1997)], the exact role of bFGF in the endogenous regulation of angiogenesis is established. Nevertheless, both observations point to the potential importance of the interaction between these two cytokines in the regulation of angiogenesis. First, it was demonstrated that VEGF and bFGF synergistically induce angiogenesis in vitro [Pepper et al., Biochem. Biophys. Res. Commun., 189: 824-831 (1992); Goto et al., Lab. Invest. 69: 508-517 (1993)], and this observation was performed in vivo in a rabbit as a model of Fuji ischemia [Asahara et al., Circulation, 92 (Supp. II): II.365-71 (1995)]. And in rats with a sponge transplant model [Hu et al., A. Br. J. Pharmacol., 114: 262-268 (1995). Second, not only its ability to induce PA activity, but also the in vitro angiogenic action of VEGF depends on endogenous bFGF produced by endothelial cells.

Alteration of endothelial function induced by VEGF family members is currently due to transmembrane tyrosine kinases including VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1) and VEGFR-3 (Flt-4). Mediated through receptors (see Mustonen et al., J. Cell Biol., 129: 895-898 (1995)). Despite the obvious deficiencies in constitutive angiogenesis, VEGFRs are expressed in many adult tissues. However, VEGFRs are clearly highly regulated in endothelial cells during development and in certain angiogenesis-related / dependent pathological situations involving tumor growth [Dvorak et al., Amer. J. Pathol., 146: 1029-1039 (1995); See Ferrara et al., Endocrine Rev., 18: 4-25 (1997). Phenotypes of VEGFR-1-deficient mice and VEGFR-2-deficient mice reveal the essential role of these receptors in angiogenesis during development. VEGFR-1-deficient mice form abnormal vascular channels due to ineffective assembly of endothelial cells into blood vessels and die intrauterine during pregnancy (Fong et al., Nature, 376: 66-70 (1995)). VEGFR-2-deficient mice die intrauterine between 8.5 and 9.5 days after mating, in contrast to VEGFR-1, possibly due to immature development of endothelial progenitors [Shalaby et al., Nature 376: 62-66]. (1995)]. The importance of VEGFR-2 in tumor angiogenesis has also been clearly demonstrated using a dominant-negative approach [Millauer et al., Nature, 367: 576-579 (1997); Millauer et al., Cancer Res. 56: 1615-1620 (1996). Phenotypes of VEGFR-3-deficient mice have not been reported yet.

Ligands for VEGFR-1 include VEGF and placental growth factor (PlGF); Ligands for VEGFR-2 include VEGF and VEGF-C; Whereas the only ligand reported so far for VEGFR-3 is VEGF-C [Mustonen et al., J. Cell Biol., 129: 895-898 (1995); Thomas, J. Biol. Chem., 271: 603-606 (1996). VEGF-C is a member of the VEGF family of angiogenic cytokines recently described. It is a protein with structural homology with VEGF, which was isolated from human prostate adenocarcinoma cell line PC-3 during the study for ligands for VEGFR-3 [Joukov et al., EMBO J., 15: 290-298 (1996) ]. Human G61 glioma cell cDNA screened with a probe based on an expression sequence tag (EST) encoding VEGF-related protein (VRP) encoding an amino acid sequence with a high degree of similarity to VEGF during an independent study for ligands against VEGFR-3 Isolated from the library [Lee et al., Proc. Natl. Acad. Sci. USA 93: 1988-92 (1996). VEGF-C and VRP are the same protein and will be referred to herein as VEGF-C. VEGF-C exhibits a high degree of similarity with VEGF and includes the conservation of eight cysteine residues involved in intramolecular and intermolecular disulfide bonds. Half of the cysteine-rich C-terminal, which increases the length of the VEGF-C / VRP polypeptide relative to other ligands of this family, exhibits a pattern of spacing cysteine residues that suggest Balbiani ring 3 protein repeats. The C-terminal propeptide also includes a short motif of the VEGF-like domain, which will promote the interaction of secreted VEGF-C with extracellular epilepsy. VEGF-C binds to the extracellular domain of VEGFR-3 and induces VEGFR-3 tyrosine phosphorylation. In addition to VEGFR-3, VEGF-C binds to induce phosphorylation of VEGFR-2. VEGF-C is less active than VEGF in this assay but promotes the growth of human and bovine endothelial cells. VEGF-C has been reported to induce endothelial cell migration in three-dimensional collagen gels (Joukov et al., EMBO J., 16: 3898-911 (1997)). VEGF-C transcripts are detectable in many adult and fetal human tissues and in many cell lines. Human VEGF-C was mapped to chromosome 4q34 (Paavonen et al., Circulation 93: 1079-82 (1996)).

One distinct feature of VEGF-C is that its mRNA is first translated into precursors that are induced to mature ligands by cell-associated proteolytic processing. After biosynthesis, VEGF-C is rapidly associated with a 58 kDa nonparallel homodimer bound by both disulfide and non-covalent bonds. Proteolytic processing of both N-terminal and C-terminal propeptide then occurs at the terminal portion of the secretory pathway and at the cell membrane, resulting in many incompletely processed intermediates. Mature VEGF-C is then released from the cell as a 21 kDa homodimer containing two VEGF-homologous domains linked by non-covalent interactions. As a result of this processing, VEGF-C acquires the ability to bind to and activate VEGFR-2 and also increases its affinity and activation properties for VEGFR-3. Intracellular proteolytic cleavage is not a prerequisite for VEGF-C secretion. Based on these observations, it has been suggested that the synthesis of VEGF-C as a precursor allows it to preferentially signal through VEGFR-3 (Joukov et al., EMBO J., 16: 3898-911 (1997)). , It is confined to the venous endothelium during early development and to the lymphatic endothelium of late and postnatal life (Kukk et al., Development, 122: 3829-3827 (1996)). It has been suggested that under certain circumstances where the processing mechanism is activated, VEGF-C gains the additional ability to signal through VEGFR-2, thereby gaining additional levels of VEGF-C activity regulation. Signaling via VEGFR requires receptor dimerization. Proteolytic processing provides another regulatory mechanism by indirectly promoting the formation of VEGFR-2 / VEGFR-3 heterodimers.

As a result of using an in vitro model of angiogenesis that analyzed for extracellular epilepsy invasion and tube formation as described by Montesano et al., Cell 42: 469-477 (1985), bFGF and VEGF resulted in small microvascular, lymph and It has been reported that aortic endothelial cells induce them to grow on three-dimensional collagen or fibrin gels to invade the underlying epilepsy that forms capillary-like tubular structures [Montesano et al., Proc. Natl. Acad. Sci. USA, 83: 7297-7301 (1986); Pepper et al., Exp. Cell. Res., 210: 298-305 (1994), Pepper et al., J. Cell. Sci., 108: 73-78 (1995). This demonstrates that angiogenic induction properties of bFGF and VEGF can be mediated through direct action on endothelial cells. However, there is increasing evidence that the nature of the response induced by a particular cytokine is determined by context, that is, by the presence or absence of other regulatory molecules in the pericellular environment of the responding cell. It has already been demonstrated that VEGF and bFGF are synergistic in an in vitro model for angiogenesis [Pepper et al., Biochem. Biophys. Res. Commun., 189: 824-831 (1992), demonstrated that TGF-β1 has a biphasic effect in the same model [Pepper et al., Exp. Cell Res., 204: 356-363 (1993).

Despite the enormous activity in the art, there remains a need for methods that affect angiogenesis. Specifically, there is a need for a method for increasing, regulating and / or inhibiting angiogenesis, and for stimulating or inhibiting the activity of proteolytic enzymes such as plasminogen activators.

1A-1F are micrographs showing angiogenic cell cord invasion of collagen gels induced by VEGF, VEGF-C, bFGF and VEGF or a combination of VEGF-C and bFGF.

2A and 2B are graphs showing in vitro angiogenesis in bovine microvascular endothelial (BME) cells induced with VEGF-C alone or in combination with bFGF.

3A and 3B are graphs showing in vitro angiogenesis in BME cells induced with VEGF-B alone or in combination with bFGF.

4A-4C are graphs showing in vitro angiogenesis in bovine aortic endothelial (BAE) cells induced by VEGF-C alone or in combination with various concentrations of bFGF.

5A and 5B are graphs showing in vitro angiogenesis in BAE cells induced with VEGF-B alone or in combination with bFGF.

6 is a graph showing in vitro angiogenesis induced by VEGF-C in bovine lymphoid endothelial (BLE) cells.

FIG. 7 is a graph showing the effect of VEGF and VEGF-C, respectively and in combination with each other on BAE cell collagen invasion.

8A and 8B are graphs showing the effect of VEGF, VEGF-B and VEGF-C, respectively, and various combinations on BAE cell collagen invasion.

9A and 9B are Zymograph analyzes of induction of tissue-type plasminogen activator (tPA) activity and urokinase-type plasminogen activator (uPA) activity, and FIG. 9C is a plasma by VEGF-C, VEGF and bFGF. Reversed-phase Zymograph analysis of Minogen Activator Inhibitor-1 (PAI-1) induction.

FIG. 10A is a Zymograph analysis of tPA activity and uPA activity induction. FIG. 10B is a reversed phase Zymograph analysis of PAI-1 activity induction by VEGF-B, VEGF and bFGF.

Figure 11 shows an increase in the quiescent level of tPA, uPA and PAI-1 mRNA induced by VEGF-C in BAE cells.

12 shows an increase in the quiescent level of uPA and PAI-1 mRNA induced by VEGF-B in BAE cells.

FIG. 13 is a graph showing the inhibition of the effect of α ₂ -antiplasmin on endothelial cell invasion of collagen gels induced by VEGF-C and bFGF in BME cells.

details

Recombinant human VEGF-B ₁₆₇ and VEGF-B ₁₈₆ can be produced as described in WO 96/26736 to Eriksson et al., Which is incorporated herein by reference.

Recombinant human VEGF-C is a baculovirus derived from the transfer plasmid pFASTBAC1 as described in Jeltsch M., Functional Analysis of VEGF-B and VEGF-C, Helsinki University (1997) using Sf9 insect cell lines. Produced in virus shuttle vector.

Recombinant human VEGF-C ΔNΔC was produced in yeast Picha pastoris (strain GS115) using the pIC9 expression vector (Invitrogen) as described by Joukov et al., EMBO J., 16: 3898-911 (1997). It became.

Recombinant human VEGF (165-amino acid homodimer species-VEGF ₁₆₅ ) was purchased from Peprotech.

Recombinant human bFGF (155 amino acid form) Provided by P. Sarmientos (Farmitalia Carlo Erba, Milan, Italy).

Bovine uPA, bovine uPAR and human tPA cDNA clones W.-D. Provided by Schleuning.

Summary of the Invention

It is an object of the present invention to characterize VEGF receptor expression in bovine endothelial cell lines.

Another object of the present invention is to develop a method for synergistically inducing angiogenic responses of endothelial cells.

Another object of the present invention is to provide a method for influencing the proteolytic characteristics of endothelial cells.

It is another object of the present invention to provide a method for inhibiting angiogenesis in endothelial cells.

Another object of the present invention is to provide a method for inhibiting the activity of a cytolytic enzyme.

It is a particular object of the present invention to provide a method for inhibiting the activity of plasminogen activator.

Yet another object of the present invention is to provide a method for inhibiting tumor growth.

Vascular endothelial growth factor-B (VEGF-B) and vasculature endothelial growth factor-C (VEGF-C) stimulate the endothelial extracellular proteolytic activity, in particular the activity of plasminogen activator, and also basic fibroblast growth Synergistic with factor (bFGF) to induce angiogenesis in endothelial cells and to regulate angiogenic activity of endothelial cells by administration or co-administration of VEGF-B and / or VEGF-C as well as other growth factors It is currently revealed.

Specifically, it has now been found that VEGF-C induces angiogenesis in endothelial cells in vitro and that both VEGF-B and VEGF-C synergize and promote angiogenesis in endothelial cells when coadministered with bFGF or VEGF.

The synergistic angiogenesis inducing action of VEGF-B or VEGF-C with bFGF can be applied not only to the regulation of physiological and pathological angiogenesis, but also to the planning of therapeutic methods for tumors and other pathological diseases.

The fact that the bovine cell line used in the following tests showed a clear response to the tested factors VEGF, VEGF-B and VEGF-C suggests that bovine receptors are sufficiently homologous to human receptors resulting in a high degree of cross-species reactivity. do. Similar or larger responses in this respect can certainly be expected in human tissues expressing human receptors.

VEGF-B and VEGF-C induce gene expression of various proteolytic enzymes, such as plasminogen activator (PA), which damages surrounding tissue and thereby facilitates the invasion of such tissue by tumor cells. Since VEGF-C can also act as a chemoattractant for endothelial cells, it can lead to invasion and penetration by lymphatic vessels (which consist of endothelial cells) and ultimately help the development of tumor metastasis. As a result, VEGF-B and VEGF-C antagonists can be used therapeutically to inhibit the action of VEGF-B and / or VEGF-C, thereby delaying or preventing permeation, endothelial cell invasion and / or metastasis. . Specifically, α2-antiplasmin may interfere with the angiogenic action of VEGF-B and / or VEGF-C as shown by Example 14 below, and therefore may be used as an anti-metastatic agent in accordance with one aspect of the present invention. .

Another aspect of the invention relates to providing an antisense nucleotide sequence complementary to at least a portion of the DNA sequence for VEGF-B and / or VEGF-C, which promotes proliferation of endothelial cells. Accordingly, the present invention provides antisense oligonucleotides that selectively bind to nucleic acid molecules encoding VEGF-B and / or VEGF-C proteins, and their use to reduce transcription and / or translation of VEGF-B or VEGF-C, respectively. It includes. This is desirable for virtually any medical condition that requires a reduction in VEGF-B and / or VEGF-C gene product expression, and that reduction is any type of tumor cell phenotype that may contribute to VEGF-B and / or VEGF-C gene expression. It includes reducing the point of view. Antisense molecules can be used in these methods to delay or retard such aspects of the tumor cell phenotype.

As used herein, the term “antisense oligonucleotide” or “antisense” describes an oligonucleotide that is an oligoribonucleotide or a modified oligodeoxyribonucleotide, which, under physiological conditions, comprises a DNA comprising a particular gene or mRNA of that gene. Hybridization with transcripts inhibits transcription of the gene and / or translation of its mRNA. Antisense molecules are designed to interfere with the transcription or translation of a target gene by hybridization with the gene. Those skilled in the art will appreciate that the exact length of the antisense oligonucleotide and the degree of complementarity with its target will depend upon the particular target selected, including that target sequence and the specific bases that the sequence includes. It is preferred that the antisense oligonucleotides are prepared and arranged to selectively bind to the target under physiological conditions, i.e., to hybridize substantially more to that target sequence than to bind to another sequence in the target cell under physiological conditions. Based on the published DNA sequences of VEGF-B and VEGF-C, those skilled in the art readily select and synthesize any of a number of suitable antisense molecules for use according to the present invention. In order to be sufficiently selective and capable for inhibition, such antisense oligonucleotides should comprise at least 7, and more preferably at least 15 consecutive bases complementary to the target [Wagner et al., Nature Biotechnology, 14: 840-844 (1996). Most preferably the antisense oligonucleotide comprises a complementary sequence of 20 to 30 bases. Oligonucleotides that are antisense are selected for any region of the gene or mRNA transcript, but in a preferred embodiment the antisense oligonucleotides correspond to N-terminal or 5 'upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions can be targeted. Targeting to mRNA splitting sites is also used in the art but is less preferred if alternative mRNA splitting occurs. In addition, the antisense sequence is preferably a site where mRNA secondary structure is not expected [Sainio et al., Cell Mol. Neurobiol., 14 (5): 439-457 (1994)] and preferably to sites where proteins are not expected to bind.

It is within the skill of the art to provide antisense nucleotides complementary to the cDNA of a gene of interest or to antisense nucleotides that hybridize with the genomic DNA of a gene of interest, because those skilled in the art will recognize that the genomic DNA as a corresponding cDNA or vice versa. Because it is easy to make. In a similar manner, antisense of alleles or homologous DNA can be obtained without undue experimentation.

Antisense oligonucleotides used in the present invention may be composed of "natural" deoxyribonucleotides, ribonucleotides or any combination thereof. The 5 'end of one natural nucleotide and the 3' end of another natural nucleotide can be covalently linked via phosphodiester internucleoside linkages as in natural systems. Such oligonucleotides can be prepared by known techniques that can be performed manually or by automated synthesizers. They can also be produced recombinantly by vectors. However, in a preferred embodiment the antisense oligonucleotides used in the present invention may also include "modified" oligonucleotides. This is that they can be modified in many ways that do not prevent them from hybridizing to their target, but it can increase their stability or targeting or otherwise increase their therapeutic effect.

As used herein, the term “modified oligonucleotide” means (1) at least two of its nucleotides are synthetic internucleoside bonds (ie, the 5 'end of one nucleotide and the 3' end of another nucleotide) Oligonucleotides in which a chemical group covalently bound to an oligonucleotide and / or (2) a chemical group that is not normally associated with a nucleic acid is described. Preferred synthetic internucleoside linkages include phosphorothioate, alkylphosphonate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate , Peptides and carboxymethyl esters. The term “modified oligonucleotide” also refers to oligonucleotides having covalently modified bases and / or sugars. For example, modified oligonucleotides include oligonucleotides having a backbone sugar covalently bonded to a lower molecular weight organic group than the hydroxyl group at the 3 'position and the phosphate group at the 5' position. Thus modified oligonucleotides comprise 2'-0-alkylated ribose groups. Furthermore, modified oligonucleotides include sugars such as arabinose instead of ribose. Modified oligonucleotides may also include base analogs such as C-5 propine modified bases (see Wagner et al., Nature Biotechnology, 14: 844 (1996)). The present invention therefore provides pharmaceutical preparations containing modified antisense molecules complementary to nucleic acids encoding VEGF-B and / or VEGF-C proteins and hybridizing with them under physiological conditions and having a pharmaceutically acceptable carrier. Come up with

Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such pharmaceutical compositions comprise antisense oligonucleotides in combination with any standard physiologically and / or pharmaceutically acceptable carrier known in the art. The composition should be sterile and should contain a therapeutically effective amount of antisense oligonucleotide in units of weight or volume suitable for administration to the patient. The term "pharmaceutically acceptable" refers to a nontoxic material that is compatible with biological systems such as cells, cell cultures, tissues or organisms. The characteristics of the carrier will vary depending on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art.

Vectors comprising such antisense sequences are used to inhibit or at least reduce the expression of the cytokine of interest. The use of this type of vector to inhibit the expression of one or more angiogenic cytokines is associated with disease, such as when the tumor produces VEGF-B and / or VEGF-C, such as to provide angiogenesis. It is beneficial if there is. Transformation of such tumor cells into a vector containing an antisense nucleotide sequence will inhibit or delay the proliferative activity of the endothelial cells, thereby inhibiting permeation, invasion and / or metastasis of the endothelial cells and consequently inhibit or delay tumor growth. .

Example 1 Cell Culture of Microvascular Endothelial Cells

Bovine adrenal cortex-induced microvascular endothelial (BME) cells (Furie et al., 1984). M.B. Obtained from Furie and SC Silverstein and supplied with minimum essential medium, 15% heat-inactivated donor calf serum (DCS) (Flow Laboratories, Baar, Switzerland), penicillin (500 U / ml) and streptomycin (100 μg / ml) Grow in alpha strain (Gibco AG, Basel, Switzerland). Cells were used at passages 16-19.

Example 2 Cell Culture of Aortic Endothelial Cells

Pepper et al., Am. J. Physiol. 262: C1246-57 (1992), as described previously in bovine aortic endothelial (BAE) cells isolated from debris of adult bovine aorta and cloned by limiting dilution, modified low glucose Dulbecco with 10% DCS and antibiotics Cultured in minimally essential medium (DMEM, Gibco). Cells were used at passages 10-15.

Example 3 Cell Culture of Lymphatic Endothelial Cells

Bovine lymphoid endothelial (BLE) cells were isolated from the lymphatic vessels of the mesentery. Provided by S. Wasi. Passage 3 cells were prepared by Pepper et al., Exp. Cloned by limiting dilution as described in Cell Res., 210: 298-305 (1994). BLE cells were cultured in DMEM with 1 mM sodium pyruvate, 10% donor calf serum and antibiotics added. Cells were used at passages 21-26.

Example 4 Cell Culture of Pulmonary Artery Endothelial Cells

Calf pulmonary endothelial (CPAE) cells were purchased from American Type Culture Collection (Rockville, MD) and cultured in medium 199 supplied with 20% DCS. Cells were used in passage 23.

All endothelial cell lines were maintained in 1.5% gelatin-coated tissue culture flasks (Falcon Labware, Becton-Dickinson Company, Lincoln Park, NJ) and subcultured at a split ratio of 1/3 or 1/4. The endothelial properties of all four cell lines have already been absorbed by DiI-Ac-LDL (Paesel and Lorei, Frankfurt, Germany) and immunostaining with rabbit polyclonal antiserum against human von Willebrand factor (Nordic Immunology, Tilburg, The Netherlands) ).

Example 5 Stimulation of Angiogenesis in BME Cells VEGF or VEGF-C Alone or in Combination with bFGF

Three-dimensional collagen gels were prepared as described by Montesano et al., Cell 42: 469-477 (1985) to measure induction of angiogenesis in vitro. Eight volumes (approximately 1.5 mg / ml) of Type I collagen solution from the rat tail cord are rapidly mixed with 1 volume of 10x minimum essential medium (Gibco) and 1 volume of sodium bicarbonate (approximately 11.76 mg / ml) on ice 18 mm tissue culture wells (Nunclon, A / S Nunc, Roskilde, Denmark) were dispensed and gelled at 37 ° C. for 10 minutes. Bovine microvascular endothelial (BME) cells were then seeded onto 500 μl medium of 0.5-1.0 × 10 ⁵ cells / well of collagen gel surface. Cells were grown and fused (3-5 days), at which point the serum was reduced to 2% and VEGF at concentrations of 30 ng / ml or VEGF-C at concentrations 0, 1, 3, 10, 30 and 100 ng / ml Or in combination with 10 ng / ml bovine fibroblast growth factor (bFGF). The medium and treatments were fresh every 2-3 days and after 4 days the medium was fixed and photographed.

Randomly selected fields of 1.0 mm x 1.4 mm were photographed in each well at a single level below the surface monolayer by a phase contrast microscope at a magnification of 105x using a Nikon Diaphot TMD reversed-phase optical microscope. 1A is a phase contrast view of an untreated control. 1B shows a sample treated with 30 ng / ml VEGF. The formation of cell cords in the resulting collagen gel is clearly evident and the focal plane is below that gel surface. 1C shows collagen gel treated with 30 ng / ml VEGF-C. 1D shows a sample treated with 10ng / ml bFGF alone. Again a clear invasive reaction can be seen. FIG. 1E shows a gel treated with a combination of 30ng / ml VEGF and 10ng / ml bFGF, and FIG. 1F shows a gel treated with a combination of 30ng / ml VEGF-C and 10ng / ml bFGF. The synergistic effects performed by co-administration of VEGF and bFGF or VEGF-C and bFGF are readily visible.

Cell cord invasion is described by Pepper et al., Biochem. Biophys. Res. As described in Commun., 189: 824-831 (1992), it is quantitatively determined by determining the total additional length of all cell structures that penetrate below the surface monolayer, either as a single cell or in the form of a cell cord. The results shown graphically in FIGS. 2A and 2B are expressed as mean total additional developmental length in μm ± sem and from at least nine photographic fields, ie from three fields from each of at least three individual experiments per condition. ¹ p <0.000, ² p <0.000 (student non-binary t-test). Mean values were compared using Student's non-binary t-test and significant p values were obtained as <0.05. For comparison, the results obtained with 30 ng / ml VEGF alone and 30 ng / ml VEGF in combination with 10 ng / ml bFGF are shown by labeled bars (VA) on the right side of each graph.

Example 6 Stimulation of Angiogenesis in BME Cells by VEGF-B in Combination with bFGF

Example 5 except that BME cell monolayers were treated with VEGF-B _{167 at} 1, 10 and 100 ng / ml concentrations and VEGF-B _{186 at} 1, 10 and 100 ng / ml concentrations alone and in combination with 10 ng / ml bFGF. The procedure of was repeated. The shorter 167 amino acid form of VEGF-B lacks the hydrophobic region and the O-glycosylation site seen in the longer 186 amino acid form. The results are shown graphically in FIG. Although VEGF-B alone did not produce measurable angiogenesis in this test, VEGF-B _{167 at} 100 ng / ml concentration and VEGF-B ₁₈₆ at all concentrations tested were combined with additional angiogenesis in 10 ng / ml bFGF. Produced more than effect. For comparison purposes, the effects of VEGF and VEGF-C alone and in combination with 10ng / ml of bFGF are shown by labeled bars of VA and VC on the right of each graph, respectively.

Example 7: Stimulation of angiogenesis in BAE cells by VEGF-C alone and in combination with bFGF

The gel surface in each well was seeded with a culture of bovine aortic endothelial (BAE) cells and tested for VEGF-C alone and in combination with bFGF at concentrations of 1 ng / ml and 10 ng / ml. The procedure was repeated again. The results are shown graphically in Figures 4 (a), 4 (b) and 4 (c), respectively. It can be seen that not only VEGF-C alone stimulates angiogenesis in BAE cells but also clearly additional angiogenic stimulatory effects are observed when the cell culture is treated with VEGF-C in combination with bFGF. Again, similar treatments with VEGF are indicated by the labeled bars (V-A) on the right side of each graph.

Example 8 Stimulation of Angiogenesis by VEGF-B in Combination with bFGF in BAE Cells

The procedure of Example 6 was repeated except that the gel surface in each well was seeded with a culture of bovine aortic endothelial (BAE) cells and bFGF was used at a concentration of 3 ng / ml. The results are shown graphically in Figures 5 (a) and 5 (b). In addition, VEGF-B alone did not produce an apparent angiogenic effect in this test, but both VEGF-B ₁₆₇ and VEGF-B ₁₈₆ produced more than an additive (ie synergistic) angiogenic effect when co-administered with bFGF. For comparison purposes, the results of VEGF (VA) and VEGF (VC) alone and in combination with bFGF are shown on the right side of each graph.

Example 9 Stimulation of Angiogenesis by VEGF-C in BLE Cells

Bovine lymphoid endothelial (BLE) cell monolayers generated by seeding on a three-dimensional gel with BLE cell culture as described in Example 5 were treated with VEGF-C and / or VEGF. Four days after treatment, randomly selected fields of the gel were photographed at a single level below the surface monolayer as described above. Endothelial cell invasion was quantified as described in connection with the above examples. The results are shown graphically in FIG. It can be seen that VEGF-C produces an apparent stimulus of angiogenic activity.

Examples 5 to 9 above show that angiogenesis-inducing properties of VEGF-B and VEGF-C can be mediated through direct action on endothelial cells and potential between VEGF-B or VEGF-C and bFGF in angiogenesis induction. It has been proven to have a synergistic effect.

The invasive response induced by VEGF-C was only measurable when added to the fusion monolayer of BME cells on three-dimensional collagen gel as shown by FIGS. 1C and 2A. In contrast, FIGS. 4A and 6 show that VEGF-C induced a clear dose dependent invasive response in BAE and BLE cells, respectively, and that of branched tube-like structures as shown by phase-control microscopy by focusing below the surface monolayer. It is accompanied by formation. VEGF-C (30 ng / ml) has slightly less ability than VEGF (30 ng / ml) because 42,000 Mr is estimated for VEGF-C when compared at equimolar (0.6-0.7 nM) concentrations. ) Was about half the capacity (Cf. 4 (a) & (b), 6 and 7).

VEGF-C induced a synergistic angiogenic response in vitro when co-administered with bFGF. Therefore, VEGF-C alone had little or no effect when added to BME cells, and VEGF-C increased the effect of bFGF up to 2.5-fold with 30 ng / ml VEGF-C as shown in FIGS. 1F and 2. Similarly, VEGF-C alone induced angiogenic activity in both BAE and BLE cells, and co-administration of VEGF-C and bFGF induced a larger additional response than shown for example in FIG. 4.

Example 10 Co-administration of VEGF and VEGF-C

Co-administration of VEGF and VEGF-C was also evaluated. ¹ p <0.000, ² p <0.000 (student non-binary t-test). Only VEGF induced invasion in BME cells and the response (measured up to 14 days) occurred for VEGF alone. In BLE cells, VEGF and VEGF-C each induced invasion and the response to co-administration of 30 ng / ml VEGF and 30 ng / ml VEGF-C was essentially additive (see Figure 6). Both VEGF and VEGF-C induced invasion in BAE cells, respectively, and the response (measured up to 4 days) was a much larger additional response than shown from the graph of FIG.

Comparisons of the effects of VEGF, VEGF-B and VEGF-C, respectively, and combinations in three ways are shown in the graphs of FIGS. 8A and 8B. 8A shows the individual effects of three cytokines. It can be seen that 30ng / ml of VEGF-B alone produces a negligible effect. VEGF (30 mg / ml) alone produced significant effects, but 30 ng / ml of VEGF-C produced the best individual effects. As can be seen from FIG. 8B, when VEGF and VEGF-B were co-administered, the result was clearly greater than the sum of their individual effects. Similarly, co-administration of VEGF-B with VEGF-C markedly increased collagen gel invasion induction activity of VEGF-C. However, the most significant effect was produced by the combination of 30 ng / ml VEGF and 30 ng / ml VEGF-C.

Example 11 Stimulation of PA and PAI-1 Activity by VEGF-C in BME and BAE Cells

Cell extracts and culture supernatants prepared from BME and BAE cells were exposed to VEGF-C at concentrations of 0, 1, 3, 10, 30 and 100 ng / ml for 15 hours and subjected to zymography and reversed-phase zymography. For comparison, test media from BME and BAE cells exposed to 30 ng / ml VEGF and 10 ng / ml bFGF, respectively, were also analyzed. The fusion monolayer of endothelial cells in a 35 mm gelatin-coated tissue culture dish was washed twice with serum free medium and each cytokine was added to serum free medium containing Trasylol (200 KIU / ml). After 15 hours, Vassalli et al., J. Exp. Med. Cell extracts were prepared as described in 159: 1653-68 (1984) and Pepper et al., J. Cell Biol., 111: 743-755 (1990) and analyzed by zymography and reversed-phase zymography.

9A shows a zymography analysis of cell extracts from BME cells. 9B shows the same zymography incubated at 37 ° C. for a longer time. 9C shows reversed-phase zymography analysis of culture supernatants from BAE cells.

Example 12 Stimulation of PA and PAI-1 by VEGF-B in BME and BAE Cells

The procedure of Example 11 was repeated except that the culture supernatants were extracted from cells exposed to 0, 1, 3, 10, 30 and 100 ng / ml of VEGF-B. The results are shown in FIGS. 10A and 10B. For comparison purposes, the results of exposure to 30 ng / ml VEGF and 10 ng / ml bFGF are also shown.

The zymography and reversed-phase zymography results shown in FIGS. 9A-9C suggest that VEGF-C induces uPA, tPA and PAI-1 activity in BME and BAE cells. Similarly, the zymography and reversed-phase zymography results shown in FIGS. 10A and 10B demonstrate that VEGF-B induces uPA and PAI-1 activity in endothelial cells. Induction of uPA and PAI-1 activity was lower than that seen with approximately equimolar concentrations of VEGF. The zymography test results also confirmed that bFGF had little or no effect on the expression of tPA. The effects of VEGF-C on PAI-1 activity in BME cells, uPA activity in BAE cells, and effects on tPA and PAI-1 in BLE cells were also tested and less significant than those shown in FIGS. 9A-9C. Turned out.

Example 13 RNA Preparation, In Vitro Transcription and Northern Blot Hybridization

Cytokines were added to the fused endothelial monolayer where fresh complete medium was added 24 hours prior. Total cell RNA was prepared after a designated time using Trizol reagent (Gibco BRL, Life Technologies, Paisley, Scotland) according to the manufacturer's instructions. Replication filter containing total cellular RNA (5 μg / lane) prepared from fusion monolayers of BME cells exposed to VEGF-C or VEGF-B (30 ng / ml) for 0, 1, 3, 9, 24 and 48 hours Were hybridized with ³² p-labeled cRNA probes. Controls are shown for periods of 0, 9, 24 and 48 hours. RNA integrity and uniformity of loading was determined by staining the filter with methylene blue after transfer and crossover as shown by 28S and 18S ribosomal RNA at the bottom of FIGS. 11 and 12. Northern blots, UV-crossing and methylene blue staining of filters, in vitro transcription, hybridization and post-hybridization washes are as previously described by Pepper et al., J. Cell Biol., 111: 743-755 (1990). [ ³² P] -labeled cRNA probes were subjected to bovine urokinase-type plasminogen activator (u-PA) [Kraetzschmar et al., Gene, 125: 177-83 (1993)], bovine u-PA receptor (u-PAR Kraetzschmar et al., Gene, 125: 177-83 (1993), human tissue-type PA (t-PA) [Fisher et al., J. Biol. Chem., 260: 11223-11230 (1985)] and bovine PA inhibitor-1 (PAI-1) [Pepper et al., J. Cell Biol., 111: 743-755 (1990)] Pepper et al., J Cell Biol., 111: 743-755 (1990) and Pepper et al., J. Cell Biol., 122: 673-684 (1993). It has been found that VEGF-C increases the stationary levels of mRNAs of uPA, tPA and PAI-1 in BAE cells.

Northern blot analysis shown in FIG. 11 suggests that VEGF-C increases the stationary levels of uPA, uPAR, tPA and PAI-1 mRNA in BAE cells. The kinetics of PA, uPAR and PAI-1 induction were fast (within 1 hour) transitive (baseline levels were obtained between 3 and 9 hours for uPA and uPAR and between 9 and 24 hours for PAI-1. ). For uPA and uPAR this is in contrast to the more encouraging increases reported for VEGF and bFGF [Pepper et al., J. Cell Biol., 111: 743-755 (1990); Mandriota et al., J. Biol. Chem., 270: 9709-16 (1995). For PAI-1 the results are similar to VEGF and bFGF. Northern blot test results for one test with VEGF-B are shown in FIG. 12.

Co-induction of this extracellular proteolytic activity, which is evident by increased synthesis of uPA, and co-induction of protease inhibition, which is evident by increased synthesis of PAI-1, is one of the demonstrations of angiogenesis both in vivo and in vitro. Consistent with the “proteolytic balance” theory, it is that while proteases are essential for cell migration and morphogenesis, protease inhibitors play an equally important acceptable role by protecting extracellular epilepsy from inappropriate destruction.

Example 16: Inhibition of endothelial angiogenesis by α ₂ -antiplasmin in the presence of VEGF-C and bFGF

BME cell monolayers were co-treated with bFGF and VEGF-C for 4 days as well as α ₂ -antiplasmin at concentrations of 0, 1, 3, 10 and 30 μg / ml. Then randomly selected fields of treated gels were photographed at a single level below the surface monolayer. Endothelial cell invasion was quantified as described above in Example 5. Figure 13 shows the results in graphical form. A clear decrease in angiogenic activity can be seen as the α ₂ -antiplasmin increasing concentration. It is therefore evident that α ₂ -antiplasmin inhibits angiogenesis induction by cotreatment with VEGF-C and bFGF in a dose-dependent manner.

The foregoing description and examples have been described for illustrative purposes only and are not intended to be limiting. Since modifications of the disclosed embodiments incorporating the concepts and substance of the invention are possible to those skilled in the art, the invention should be construed to encompass all that is within the scope of the appended claims and their equivalents.

references

The following documents provide the technical background of the present invention and are specifically incorporated by reference in this application.

Claims (25)

A method of stimulating angiogenesis in endothelial cells, the method comprising co-administering said cells with at least two cytokines selected from the group consisting of VEGF, VEGF-B, VEGF-C, and FGF. The method of claim 1, wherein the endothelial cells are selected from the group consisting of microvascular endothelial cells, aortic endothelial cells and lymphatic endothelial cells. The method of claim 2, wherein the endothelial cells are bovine endothelial cells. The method of claim 2, wherein the endothelial cells are human endothelial cells. 2. The method of claim 1, wherein said cells are treated by in vitro culture in a culture medium and cytokines are administered by integrating them into said culture medium. The method of claim 1, wherein the cytokine is administered in the form of purified protein. The method of claim 1, wherein the cytokine is administered by transfecting or transforming endothelial cells with at least one vector comprising a cytokine encoding nucleotide sequence operably linked to at least one promoter sequence. The method of claim 1, wherein at least two cytokines comprise VEGF-C and FGF. The method of claim 1, wherein at least two cytokines comprise VEGF-B and FGF. The method of claim 1, wherein the at least two cytokines comprise VEGF and VEGF-C. The method of claim 1, wherein the at least two cytokines comprise VEGF-B and VEGF-C. The method of claim 1, wherein at least two cytokines comprise VEGF and VEGF-B. A method of regulating endothelial angiogenesis by cells releasing a synergistic combination of at least two angiogenic cytokines selected from the group consisting of VEGF, VEGF-B, VEGF-C and FGF, said method comprising Neutralizing one of the dog's angiogenic cytokines. The method of claim 13, wherein said neutralization is performed by treating the cells with an antibody against an angiogenic cytokine being neutralized. 15. The method of claim 14, wherein the neutralized cytokine is VEGF-C. The method of claim 14, wherein the neutralized cytokine is VEGF-B. The method of claim 14, wherein said antibody is a monoclonal antibody. The method of claim 13, wherein said cells are solid tumor cells. A method of inhibiting angiogenesis by endothelial cells in the presence of VEGF-C and FGF, the method comprising treating the cells with antiplasmin. 20. The method of claim 19, wherein said antiplasmin comprises α ₂ -antiplasmin. 20. The method of claim 19, wherein said endothelial cells are selected from the group consisting of microvascular endothelial cells, aortic endothelial cells and lymphatic endothelial cells. A method of stimulating proteolytic activity of endothelial cells, the method comprising: treating cells with VEGF-B or VEGF-C or a combination of VEGF-B or VEGF-C with at least one additional cytokine selected from VEGF and FGF How to include. 23. The method of claim 22, wherein said endothelial cells are selected from the group consisting of microvascular endothelial cells, aortic endothelial cells and lymphatic endothelial cells. A method of inhibiting endothelial cell permeation, invasion and / or metastasis of a patient, the method comprising administering to said patient an effective amount of VEGF-B or VEGF-C antagonist at an endothelial cell proliferation inhibition. A method of modulating angiogenic activity of endothelial cells, the method comprising transfecting or transforming a cell with a vector containing antisense nucleic acid for VEGF-B or VEGF-C.