JP2001514867A - Stimulation, regulation and / or inhibition of endothelial proteolytic and / or angiogenic activity - Google Patents

Abstract

  (57) [Summary] Vascular endothelial growth factor-B (VEGF-B) and vascular endothelial growth factor-C (VEGF-C) are angiogenic polypeptides. VEGF-B and -C have been shown to be angiogenic in vitro, especially when combined with bFGF. VEGF-C also increases plasminogen activator (PA) activity in bovine endothelial cell lines, and is accompanied by a concomitant increase in PA inhibitor-1. Addition of α-2-antiplasmin (antiplasmin) to bovine endothelial cells co-treated with bFGF and VEGF-C partially inhibits entry into collagen gel.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Angiogenesis is the formation of new capillaries by the process of sprouting out of existing blood vessels, occurring in development and in many physiological and pathological regulations. Angiogenesis is therefore necessary for tissue growth, wound healing, and female reproductive function, and is also a component of pathological processes such as tumor growth, hemangiogenesis and ocular neovascularization (Folkman (Folkman), New Engl. J
Med., 333: 1757-1763 (1995); Pepper, Arterioscler. Thromb.
Vasc. Biol., 17: 605-619 (1997)). A similar, but poorly studied process occurs in the lymphatic system, which involves lymphangiogenesis (lym
phangiogenesis).

Angiogenesis is initiated by the localized breakdown (degradation) of the basement membrane of the parent vessel, followed by migration and proliferation of endothelial cells into the surrounding extracellular matrix, As a result, capillary sprouting is formed. Subsequently, the lumen is formed and constitutes an essential element in functional sprouting. Sprout maturation is completed by reconstitution of the basement membrane. During this series of events, at least three changes in the function of endothelial cells occur: 1) modulation of the interaction with the extracellular matrix, which involves changes in cell-substrate (matrix) contact and substrate-degradable proteins. Degrading enzyme (
Requires the production of plasminogen activator (PA) and a substrate (matrix) metalloproteinase); 2) an initial increase and subsequent decrease in locomotion (migration), which causes cells to roll toward angiogenic stimuli. Position and stop when cells reach their destination; 3) increased proliferation, which supplies new cells for blood vessel growth and elongation, and becomes quiescent when blood vessels are subsequently formed Return to The function of these cells also contributes to the process of capillary morphogenesis, the formation of three-dimensional patency or open tube-like structures. Many newly formed capillaries subsequently undergo a process of vessel wall maturation (ie, the formation of media and adventitia rich in smooth muscle cells) and regression (ie, absence of blood flow). (See Pepper et al., Enzyme Protein, 49: 138-162 (1996); Risau, Nature 386: 671-674 (1997)).

[0003] Usually, with the exception of angiogenesis that occurs in response to tissue damage or in the female reproductive tract, the turnover of endothelial cells in healthy adult organisms is said to be very low. The maintenance of endothelial quiescence is thought to be due to the presence of endogenous negative regulators. This is because positive regulators are frequently detected in adult tissues where apparently no neovascularization has occurred. The converse is also true. That is, in a tissue where endothelial cell turnover is increased, both a positive regulator and a negative regulator are often present. This allows the "angiogenic switch (ang
The concept of "iogenic switch)" is derived from the concept that the activation state of the endothelium is determined by the balance between the positive and negative regulators. In activated (angiogenic) endothelium, Positive regulators predominate, while endothelial quiescence is achieved and maintained by dominance of negative regulators (Hanahan et al.,
Cell, 86: 353-364 (1996)). Initially, in tumor progression, prevasculature (prevasc
ular) stage was used to describe the transition from the vascular stage to the vascular stage.
The concept of "switch" has also been applied in developmental, physiological, and pathological angiogenesis, which has not yet been proven conclusively in vivo, but the current research hypothesis states: The switch is said to be involved in inducing positive regulators and / or reducing negative regulators.

[0004] Many polypeptide growth factors or cytokines are angiogenic (ang
iogenic) (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 include vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, and basic fibroblast growth factor (bFGF). However, the role of VEGF in the development of embryonic vasculature and in tumor angiogenesis has been clearly demonstrated (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., End.
ocrine Rev., 18: 4-25 (1997)), but the exact role of bFGF in the intrinsic regulation of angiogenesis has not yet been proven. Nevertheless, 2
Two findings point to the potential importance of the interaction between these two cytokines in regulating angiogenesis. First, VEGF and bFGF have been shown to cooperate in inducing angiogenesis in vitro (Pepper (Pe
pper) et al., Biochem. Biophys. Res. Commun., 189: 824-831 (1992);
to) et al., Lab. Invest. 69: 508-517 (1993)) and this finding has been demonstrated in rabbits in a hindlimb ischemia model (Asahara et al., Circulation, 92 (Supp. II): II.
65-71 (1995)), and in rats with a corpus cavernosum implant model (Hu
Et al., A. Br. J. Pharmacol., 114: 262-268 (1995)). Second, both the angiogenic effect of VEGF in vitro and its ability to induce PA activity are dependent on endogenous bFGF produced by endothelial cells.

[0005] Alterations in endothelial cell function induced by members of the VEGF family are now VEGFR-1 (Flt-1), VEGFR-2 (KDR / F
lk-1) and VEGFR-3 (Flt-4), including those mediated by transmembrane tyrosine kinase receptors (Mustonen et al., J. Cell Biol.
., 129: 895-898 (1995)). VEGFR is expressed in many adult tissues despite apparently no constitutive neovascularization. But VE
GFR is clearly upregulated in endothelial cells in certain angiogenic-related / dependent pathological situations, including during development and tumor growth (Dvorak et al., Amer. J. Pathol., 146: 1029-1039 (1995); Farrara (Fe
rrara) et al., Endocrine Rev., 18: 4-25 (1997)). The phenotype of VEGFR-1-deficient and VEGFR-2-deficient mice revealed an important role for these receptors in angiogenesis during development. VEGFR-1-deficient mice died in the uterus during mid-gestation due to the formation of abnormal vascular channels as a result of inefficient assembly of endothelial cells into blood vessels (assembly). Fong et al., Nature, 376: 66-70 (1995)). VEGFR-2-deficient mice die in utero between 8.5 and 9.5 days after copulation, which results in VEGFR-deficient mice.
Unlike in case 1, it appeared to be due to an undergrowth of endothelial cell precursors (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 (1994); Millauer et al., Cancer Res. 56: 1615-1620 (1996)). The phenotype of VEGFR-3-deficient mice has not yet been reported.

[0006] Ligands for VEGFR-1 include VEGF and placental growth factor (PlGF)
Ligands for VEGFR-2 include VEGF and VEGF-C; whereas, the only ligand so far reported for VEGFR-3 is VEGF-C (Mustonen et al. , J. Cell Biol., 129: 8.
95-898 (1995); Thomas, J. Biol. Chem., 271: 603-606 (1996)). VEGF-C is a recently reported member of the VEGF family of angiogenic cytokines. It is a protein that shows structural homology to VEGF and was isolated from the human prostate cancer cell line PC-3 while searching for a ligand for VEGFR-3 (Joukov et al., EMBO J., 15: 290-298 (1996)
See). In a separate search for ligands for VEGFR-3, V
EGF-related protein (VRP) was isolated from a human G61 glioma cell cDNA library. This library was screened with a probe based on the expressed sequence tag (EST) encoding an amino acid sequence with a high degree of similarity to VEGF (Lee et al., Proc. Natl. Acad. Sci. USA 93: 198).
8-92 (1996)). VEGF-C and VRP are the same protein, and are hereinafter referred to as VEGF-C. VEGF-C shows a high degree of similarity to VEGF, conserving eight cysteine residues involved in intra- and intermolecular disulfide bonds. By the cysteine-rich C-terminal half, VEGF-C /
VRP polypeptides are longer in length compared to other ligands in this family, indicating a pattern of cysteine residue spacing reminiscent of the Balbiani ring 3 protein repeat. The C-terminal propeptide also contains a short motif of the VEGF-like domain, which may facilitate the interaction of secreted VEGF-C with the extracellular matrix. VEGF-C is VEGFR
-3, which induces tyrosine phosphorylation of VEGFR-3. In addition to VEGFR-3, VEGF-C also binds to VEGFR-2 and induces its phosphorylation. VEGF-C has VEGF activity in this assay.
Lower than GF, but promotes proliferation of human and bovine endothelial cells. VEGF-C
Induces the migration of endothelial cells in a three-dimensional collagen gel (Joukov et al., EMBO J., 16: 3898-911 (1977)
)). VEGF-C transcripts are detectable in many adult and fetal human tissues and in many cell lines. Human VEGF-C has been mapped to chromosome 4q34 (Paavonen et al., Circulation 93: 1079-82 (1996)).

One of the striking features of VEGF-C is that its mRNA is first translated into a precursor, and mature ligands from that precursor are generated by cell-associated proteolytic processing. . After biosynthesis, VEGF-C
Rapidly associate into a 58 kDa antiparallel, homodimer linked by both disulfide and non-covalent bonds. This is followed by proteolytic processing of both the N- and C-termini of the propeptide, at the end of the secretory pathway and at the cell membrane, resulting in a number of incompletely processed intermediates. The mature VEGF-C is then released from the cell as a 21 kDa homodimer containing two VEGF-homology domains bound by non-covalent interactions. As a result of this processing, VEGF-C
Gains the ability to bind and activate EGFR-2 and
Increases its affinity and activation properties for R-3. Intracellular proteolytic cleavage is not essential for VEGF-C secretion. Based on these findings, VEGF-C is synthesized as a precursor,
It has been suggested that it allows for preferential signaling via FR-3 (Joukov et al., EMBO J., 16: 3898-911 (1997)). VEGFR-3 is restricted to venous endothelium early in development and becomes lymphoid endothelium later in development and in postnatal life (Kukk et al.,
Development, 122: 3829-3827 (1996)). In certain situations, such as when the processing mechanism is activated, VEGF-C gains the additional ability to signal via VEGFR-2, thereby increasing the level of regulation of VEGF-C activity. It has been suggested that it may be provided. Signaling via VEGFR requires receptor dimerization. Proteolytic processing may provide an additional regulatory mechanism by indirectly promoting the formation of VEGFR-2 / VEGFR-3 heterodimers.

[0008] By using an in vitro angiogenesis model that assayes for extracellular matrix invasion and tube formation as described by Montesano et al., Cell 42: 469-477 (1985). And VEGF induce the invasion of bovine microvessels, lymph, and aortic endothelial cells grown on three-dimensional collagen or fibrin gel into the underlying matrix (matrix). It has been reported that these endothelial cells form a capillary-like tubular structure (Montesano et al., Proc. Natl. Acad. Sci. USA, 8).
3: 7297-7301 (1986); Pepper et al., Exp. Cell Res., 210: 298-305 (1
994); Pepper et al., J. Cell Sci., 108: 73-78 (1995)). This indicates that the angiogenic properties of bFGF and VEGF can be mediated by direct effects on endothelial cells. However, there is evidence that the nature of the response elicited by a specific cytokine is contextual, i.e., dependent on the presence or absence of other regulatory molecules in the pericellular environment of the responding cell. Has been gained. For angiogenesis, VEGF and bFGF cooperate in an in vitro model (Pepper).
) Et al., Biochem. Biophys. Res. Commun., 189: 824-831 (1992)) and that TGF-β1 has a biphasic effect in the same model (Pepper (Pe
pper) et al., Exp. Cell Res., 204: 356-363 (1993)) have been previously shown.

[0009] Despite the intensive activities in the art, there remains a need for methods of affecting angiogenesis. In particular, there is a need for methods of enhancing, modulating and / or inhibiting angiogenesis and for stimulating or inhibiting the activity of proteolytic enzymes such as plasminogen activator.

SUMMARY OF THE INVENTION It is an object of the present invention to characterize VEGF receptor expression in a bovine endothelial cell line.

[0011] Another object of the invention is to synergistically (synergi) the endothelial cell angiogenic response.
stically)) to develop a way to guide.

It is a further object of the present invention to provide a method for affecting the proteolytic nature of endothelial cells.

It is also an object of the present invention to provide a method for suppressing an angiogenic response in endothelial cells.

Yet another object of the present invention is to provide a method for inhibiting the activity of a proteolytic enzyme.

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

[0016] Yet another object of the present invention is to provide a method for inhibiting tumor growth.

In the present invention, vascular endothelial growth factor-B (VEGF-B) and vascular endothelial growth factor-C (VEGF-C) exert an extracellular proteolytic activity of endothelium, in particular, an activity of plasminogen activator. Stimulating and also inducing angiogenesis in endothelial cells in cooperation with basic fibroblast growth factor (bFGF); and
It has been found that administration or co-administration of VEGF-B and / or VEGF-C and other growth factors can modulate the angiogenic activity of endothelial cells.

In particular, in the present invention, VEGF-C induces angiogenesis in endothelial cells in vitro, and both VEGF-B and VEGF-C were co-administered with bFGF or VEGF. In some cases, it was found that they synergistically (synergistically) promote angiogenesis in endothelial cells.

The synergistic (synergistic (syn) of VEGF-B or VEGF-C and bFGF
ergistic)) Angiogenic effects may be applicable in the regulation of physiological and pathological angiogenesis, and in the design of therapeutic strategies for tumors and other pathological conditions.

The bovine cell line used in the following tests was human VEGF, VEGF-
The fact that it shows a clear response to B and VEGF-C indicates that the bovine receptor is sufficiently homologous to the human receptor to produce a high degree of cross-reactivity between species. Is shown. In this regard, a similar or greater response can be expected in human tissues expressing human receptors.

VEGF-B and VEGF-C are plasminogen activators (PA
) To induce gene expression of various proteolytic enzymes. Proteolytic enzymes can damage surrounding tissues, thereby promoting the invasion of tumor cells by those cells. VEGF-C is a chemoattractant for endothelial cells (
This can act as a chemoattractant, which results in lymphatic vessels (
Lymphatic vessels can also direct invasion and penetration by endothelial cells) and may ultimately assist in the initiation (onset) of tumor metastasis. Therefore, VEGF-B and VEGF-C antagonists are VEGF-B
And / or can be used therapeutically to inhibit the action of VEGF-C, and thereby delay or prevent penetration, endothelial cell invasion and / or metastasis. In particular, as shown in Example 14 below, α2-antiplasmin can block the angiogenic effect of VEGF-B and / or VEGF-C (react with the angiogenic effect), and thus provide one of the present invention. Side view,
It can be used as an anti-metastatic agent.

Another aspect of the present invention is to provide an antisense nucleotide sequence complementary to at least a portion of a DNA sequence for VEGF-B and / or VEGF-C that promotes endothelial cell proliferation. . The present invention therefore relates to VEGF
Antisense oligonucleotides that selectively bind to nucleic acid molecules encoding B- and / or VEGF-C proteins, and VEGF-B or VEGF
-C genes, including their use to reduce transcription and / or translation. This is preferred in virtually any medical setting where it is desired to reduce VEGF-B and / or VEGF-C gene product expression. This is VEGF
-Reducing any aspect of the tumor cell phenotype that may be due to B and / or VEGF-C gene expression. Antisense molecules can be utilized in such a way as to delay or arrest aspects of such tumor cell phenotypes.

As used herein, the term “antisense oligonucleotide” or “antisense” hybridizes under physiological conditions to DNA consisting of a particular gene or mRNA transcript of that gene. And / or modified oligodeoxyribonucleotides, thereby inhibiting the transcription of the gene and / or the translation of the mRNA. Antisense molecules are designed to interfere with transcription or translation of a target gene by hybridization with the target gene. One skilled in the art will appreciate that the exact length of the antisense oligonucleotide and its degree of complementarity with the target will depend on the particular target chosen, including the target sequence and the specific bases making up the target sequence. You will recognize that there will be. An antisense oligonucleotide is capable of selectively binding to its target under physiological conditions,
That is, it is preferably constructed and designed to hybridize substantially better to the target sequence than other sequences in the target cell under physiological conditions. VEGF-
Based on the published DNA sequences of B and VEGF-C, those skilled in the art will be able to readily select and synthesize any of a number of suitable antisense molecules for use in accordance with the present invention. To be sufficiently selective and potent to inhibit, such antisense oligonucleotides should have at least 7,
More preferably it should consist of at least 15 consecutive bases complementary to the target (Wagner et al., Nature Biotechnology, 14: 840-8).
44 (1996)). Most preferably, the antisense oligonucleotide consists of a complementary sequence of 20 to 30 bases. The oligonucleotide is a gene or mR
Although may be selected from those that are antisense to any region of the NA transcript, in preferred embodiments, the antisense oligonucleotide comprises a translation initiation site, a transcription initiation site, or a promoter site. Those corresponding to the N-terminal or 5 'upstream site are desirable. In addition, the 3'-untranslated region may be targeted. Targeting to mRNA splicing sites has also been used in the art, but is less preferred where alternative mRNA splicing occurs. Furthermore, the antisense sequence may be located at a site where mRNA secondary structure is not expected to occur (Sainio et al., Cell Mol. Neurobiol., 14 (5
): 439-457 (1994)), and preferably targeting sites where the protein is not expected to bind.

It is believed that those skilled in the art can provide an antisense nucleotide complementary to the cDNA of the desired gene or an antisense nucleotide that hybridizes to the genomic DNA of the desired gene. For a person skilled in the art would easily be able to derive the genomic DNA corresponding to the cDNA and vice versa. Similarly, antisense to allelic or homologous DNA could be obtained without undue experimentation.

The antisense oligonucleotide used in the present invention is a “natural (nat)
ural) "can be composed of deoxyribonucleotides, ribonucleotides, or a combination thereof, that is, it is one natural (nat)
ive) The 5 'end of a nucleotide and the 3' end of another native nucleotide may be covalently linked by a phosphodiester internucleoside linkage in a natural system. it can. These oligonucleotides can be prepared by methods well known to those skilled in the art, which may be performed manually or by an automatic synthesizer. The oligonucleotides may be produced recombinantly using a vector. However, in preferred embodiments, the antisense oligonucleotides used in the present invention may also include "modified" oligonucleotides. That is, the oligonucleotides are modified in a number of ways that do not prevent them from hybridizing to the target and that enhance their stability or targeting, in other words, enhance their therapeutic efficacy. You may.

As used herein, the term “modified oligonucleotide” refers to (1) at least two nucleotides of which are synthetic internucleoside linkages (ie, the 5 ′ end of one nucleotide and the other Oligonucleotides covalently linked by a linkage other than a phosphodiester bond to the 3 'end of two nucleotides, and / or (2) chemical groups that are not normally linked to nucleic acids Refers to an oligonucleotide that is covalently linked. Preferred synthetic internucleoside linkages include phosphorothiates (phosphorothiates).
ioate), alkyl phosphonate (alklphosphonate), phosphorodithioate (phos
phorodithioate), phosphate ester, alkylphosphonothioate, phosphoramidite
e), carbamate, carbonate, phosphate (phosp
hate) triesters, acetamidodates, peptides, and carboxymethyl esters. The term "modified oligonucleotide" also includes oligonucleotides comprising covalently modified bases and / or sugars. For example, the modified oligonucleotide has its 3 ′
A backbone sugar in which a low molecular weight organic group which is not a hydroxyl group at the position and a low molecular weight organic group which is not a phosphate group are covalently bonded at the 5 ′ position.
and s). Thus, the modified oligonucleotide may contain a 2'-O-alkylated ribose group. Further, the modified oligonucleotide may contain a sugar such as arabinose instead of ribose. Modified oligonucleotides can also include C-5 propyne (propy).
ne) may contain base analogs such as modified bases (Wagner et al.,
Nature Biotechnology, 14: 844 (1996)). The present invention therefore relates to VE
A modified antisense molecule that is complementary to a nucleic acid encoding a GF-B and / or VEGF-C protein and that hybridizes under physiological conditions, together with a pharmaceutically acceptable carrier , Intended for pharmaceutical preparations.

[0027] The antisense oligonucleotide may be administered as part of a pharmaceutical composition. Such pharmaceutical compositions may include the antisense oligonucleotide in any of the standard physiological and / or pharmaceutically acceptable carriers known to those of skill in the art. The composition must be sterile and should contain a therapeutically effective amount of the antisense oligonucleotide in units of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interfere with the effectiveness of the biological activity of the principal component. “Physiologically acceptable (p
The term "hysiologically acceptable" refers to non-toxic substances that are compatible with biological systems, such as cells, cell cultures, tissues or organisms. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, excipients, salts, buffers, stabilizers, solubilizers, and other materials well known to those skilled in the art.

Vectors containing such antisense sequences can be used to inhibit, or at least attenuate, the expression of the relevant cytokine. The use of this type of vector to inhibit the expression of one or more angiogenic cytokines, such as when the tumor produces VEGF-B and / or VEGF-C to provide angiogenesis It may be advantageous when the expression of the cytokine is associated with a disease. Transformation of such tumor cells with a vector containing an antisense nucleotide sequence may suppress or slow the proliferative activity of endothelial cells, thereby allowing penetration, endothelial invasion (invasion). And / or metastasis could be inhibited, resulting in inhibition or delay of tumor growth.

EXAMPLES Detailed Description Recombinant human VEGF-B ₁₆₇ and VEGF-B ₁₈₆ are disclosed in Eriksson et al., WO 96/2673, the disclosure of which is incorporated herein by reference.
6 can be produced.

[0030] Recombinant human VEGF-C is derived from the Sf9 insect cell line (National Public Health Inst.
itute, Helsinki) and Jeltsch M., Functional Analys
Produced in a baculovirus system using a baculovirus shuttle vector derived from the transfer plasmid pFASTBAC1 described in is of VEGF-B and VEGF-C, Helsinki University (1997).

Recombinant human VEGF-C ΔNΔC was obtained from Joukov et al., EMBO J., 16:
PIC9 expression vector (Invitrogen) as described by 3898-911 (1997).
Was produced in the yeast Pichia pastoris (strain GS115).

Recombinant human VEGF (a species of 165 amino acid homodimer—VEGF ₁₆₅ )
Purchased from eprotech.

[0033] Recombinant human bFGF (155 amino acid form) was provided by Dr. P. Sarmientos (Farmitalia Carlo Erba, Milan, Italy).

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

Example 1 Cell Culture of Microvascular Endothelial Cells Bovine adrenal cortex-derived microvascular endothelial (BME) cells (Furie et al., 1)
984) was obtained from Dr. MB Furie and Dr. S. C. Silverstein and obtained from 15% heat-inactivated donor bovine serum (DCS). (Flow Laboratories, Baar, Switzerland)
, Penicillin (500 U / ml) and streptomycin (100 μg / ml)
) Were added and grown in minimal basal medium, α-corrected (Gibco AG, Basel, Switzerland). Cells between passages 16 and 19 were used.

Example 2: Cell culture of aortic endothelial cells [0036] Adult bovine thoracic aorta scraping as previously described in Pepper et al., Am. J. Physiol. 262: C1246-57 (1992). Bovine aortic endothelial (BAE) cells, isolated by limiting dilution, were isolated from 10% D
Low glucose Dulbecco's modified minimal basal medium supplemented with CS and antibiotics (DM
EM, Gibco). Cells between passages 10 and 15 were used.

Example 3 Cell Culture of Lymphatic Endothelial Cells Bovine lymphatic endothelial (BLE) cells were isolated from mesenteric lymphatic vessels and provided by Dr. S. Wasi. Passage 3 cells were cloned by limiting dilution as described by Pepper et al., Exp. Cell Res., 210: 298-305 (1994). BLE cells were treated with 1 mM sodium pyruvate (s
odium pyruvate), DMEM supplemented with 10% donor bovine serum and antibiotics. Cells between passages 21 and 26 were used.

Example 4: Cell culture of pulmonary artery endothelial cells Calf pulmonary artery endothelial (CPAE) cells were collected from the American Type Culture Collection (
Rockville, MD) and cultured in Medium 199 supplemented with 20% DCS. Cells from passage 23 were used.

[0039] All endothelial cell lines were maintained in 1.5% gelatin-coated tissue culture flasks (Falcon Labware, Becton-Dickinson Company, Lincoln Park, NJ) and split at 1/3 or 1/4. And subcultured (cultured). The endothelial properties of all four cell lines were determined by incorporation of DiI-Ac-LDL (Paesel and Lorei, Frankfurt, Germany) and human von Willebrand factor (Nordic Immunology, Tilburg, Theil). It was previously confirmed by immunostaining with rabbit polyclonal antiserum to Netherlands).

Example 5: Stimulation of angiogenesis in BME cells by VEGF or VEGF-C alone or in combination with bFGF To determine the induction of angiogenesis in vitro, Montesano et al., C
Three-dimensional collagen gels were prepared as described in ell 42: 469-477 (1985). 8 of a solution of type I collagen (about 1.5 mg / ml) from rat tail tendon
The volume was quickly mixed on ice with one volume of 10x minimal basal medium (Gibco) and one volume of sodium bicarbonate (11.76 mg / ml), and mixed in an 18 mm tissue culture well.
(Nunclon, A / S Nunc, Roskilde, Denmark) and allowed to gel at 37 ° C. for 10 minutes. Next, bovine microvascular endothelial (BME) cells were seeded on the surface of the collagen gel at 0.5-1.0 × 10 ⁵ cells / well in 500 μl of medium. The cells are allowed to grow to confluence (3-5 days), at which point the serum is reduced to 2% and the cell monolayer is grown at a concentration of 30 ng / ml VEGF or 0,1,3,
VEGF-C alone at concentrations of 10, 30 and 100 ng / ml and 10
Treated in combination with ng / ml bovine fibroblast growth factor (bFGF). Medium and treatments were renewed every 2-3 days and cultures were fixed and photographed 4 days later.

A randomly selected area of 1.0 mm × 1.4 mm was used for Nikon Diapho
A single level below the surface monolayer in each well was photographed by phase contrast microscopy at 105x magnification using a tTMD inverted microscope photographer. FIG. 1a is a phase contrast view of the untreated control. FIG. 1b shows a sample treated with 30 ng / ml VEGF. As a result, it can be clearly seen that a cell cord was formed in the collagen gel. The plane of focus is below the gel surface. FIG. 1c shows a collagen gel treated with 30 ng / ml VEGF-C. FIG. 1d shows a sample treated with 10 ng / ml bFGF alone. Again, a clear intrusion response is seen. FIG.
FIG. 1f shows a gel treated with a combination of ng / ml VEGF and 10 ng / ml bFGF, and FIG. 1f shows a gel treated with a combination of 30 ng / ml VEGF-C and 10 ng / ml bFGF. The synergistic effect achieved by co-administration of VEGF and bFGF or co-administration of VEGF-C and bFGF is readily seen.

To measure the total additive length of all cellular structures that have penetrated beneath a surface monolayer, as apparently as a single cell or in the form of a cell cord, invasion of the cell cord. By Pepper et al., Biochem. Biophys. Res. Commun.
, 189: 824-831 (1992). The results shown graphically in FIGS. 2a and 2b are in μm ± s. e. m. Mean total sprouts (total
expressed as additive sprout length, which is from at least nine regions of the photograph, ie, three regions from each of at least three separate experiments per condition. ¹ P <0.000, ² P <0.000 (Student's unpaired t-test). Means were compared using Student's unpaired test, with significant P values <0.05. For comparison purposes, on the right side of each graph, the results obtained with 30 ng / ml VEGF alone and the combination of 30 ng / ml VEGF and 10 ng / ml bFGF are shown in (VA)
Is indicated by a bar labeled.

Example 6: Combination of VEGF-B and bFGF stimulates angiogenesis in BME cells VEGF-B _{167 at} a concentration of 1, 10 and 100 ng / ml and VEGF at a concentration of 1, 10 and 100 ng / ml -B ₁₈₆ alone, and 10 ng / ml except for processing the BME cell monolayers by the combination of bFGF in, the same procedure was repeated as in example 5. The shorter 167 amino acid form of VEGF-B lacks the hydrophobic region and O-glycosylation site found in the longer 186 amino acid form. The results are shown graphically in FIG. VEGF-B alone did not produce measurable angiogenesis in this test, but 10 ng / ml bF
When combined with GF, VEGF-B _{167 at} a concentration of 100 ng / ml and VEGF-B _{186 at} all concentrations tested produced more vascular neoplasms than additive effects. For comparison purposes, the right side of each graph shows VEGF and VEGF-
The effects of administration of C alone and in combination with 10 ng / ml bFGF are indicated by bars labeled VA and VC, respectively.

Example 7: Stimulation of angiogenesis in BAE cells by VEGF-C alone and in combination with bFGF A culture of bovine aortic endothelial (BAE) cells was seeded on the surface of a gel in each well and VEGF-C alone And the same procedure as in Example 5 was repeated, except that it was tested in combination with bFGF at concentrations of 1 ng / ml and 10 ng / ml. The results are shown graphically in FIGS. 4 (a), 4 (b) and 4 (c) respectively. VEGF-C alone not only stimulated angiogenesis in BAE cells, but when cell cultures were treated with a combination of VEGF-C and bFGF, a greater effect than the apparent additive angiogenic stimulation was observed. It is understood that it was done. Again, to the right of each graph, the results of comparable comparisons with VEGF are shown by bars labeled (VA).

Example 8: Combination of VEGF-B and bFGF stimulates angiogenesis in BAE cells Bovine aortic endothelial (BAE) cell cultures are seeded on the surface of the gel in each well and bFGF at a concentration of 3 ng / ml The same procedure as in Example 6 was repeated, except that was used. The results are shown graphically in FIGS. 5 (a) and 5 (b). Again, VEGF-B alone had no apparent angiogenic effect in this test, but when co-administered with bFGF, both VEGF-B ₁₆₇ and VEGF-B ₁₈₆ showed more than an additive effect. A large (ie, synergistic) angiogenic effect was produced. For comparison purposes, the results achieved with VEGF (VA) and VEGF-C (VC) alone and in combination with bFGF are shown on the right side of each graph.

Example 9: Stimulation of angiogenesis in BLE cells with VEGF-C Bovine lymphatic endothelial (BLE) cell monolayers formed by seeding a BLE cell culture on the three-dimensional gel described in Example 5 , VEGF-C and / or VEGF. Four days after treatment, randomly selected gel areas were
As before, images were taken at a single level below the surface monolayer. Endothelial cell invasion was quantified as described in the combination of the previous examples. The results are shown graphically in FIG. It is understood that VEGF-C caused a distinct stimulation of angiogenic activity.

Examples 5 to 9 above show that the angiogenic properties of VEGF-B and VEGF-C can be mediated by a direct effect on endothelial cells;
It demonstrates that there is a strong synergistic effect between VEGF-B or VEGF-C and bFGF in inducing angiogenesis.

As shown by FIGS. 1c and 2a, BME on three-dimensional collagen gel
When added to a confluent monolayer of cells, the invasive response induced by VEGF-C was hardly measurable. In contrast, FIGS. 4a and 6 show that VEGF-C induces a distinct dose-dependent invasive response in BAE and BLE cells, respectively, with concomitant phase shift by focusing below the surface monolayer. As can be seen by microscopy, a branched tube-like structure has been formed. The VEGF-C believe M _r 42,000, as compared with the case of the concentration of the equimolar (0 .6-0.7nM), VEGF-C (30ng / ml
) Is slightly weaker than VEGF (30 ng / ml) and bFGF (10 ng / ml).
ml) (see FIGS. 4 (a) & (b), 6 and 7).

When co-administered with bFGF, VEGF-C induced a synergistic in vitro angiogenic response. That is, VEGF-C alone had little or no effect when added to BME cells, but as shown in FIGS.
Enhances the effect of bFGF, up to 2.5 at 30 ng / ml VEGF-C.
Fold increase. Similarly, in both BAE and BLE cells, VEG
Although FC alone induces angiogenic activity, for example, as shown in FIG.
When -C and bFGF were co-administered, they induced a greater response than the additive response.

Example 10: Co-administration of VEGF and VEGF-C The effect of co-addition of VEGF and VEGF-C was also evaluated. ¹ P <0
. 000, ² P <0.000 (Student's unpaired test
t-test)). In BME cells in which VEGF alone induced invasion, a response (measured by day 14) occurred for VEGF alone. VEGF and VEGF-
In BLE cells, each of which individually induced invasion, the response to co-administration of 30 ng / ml VEGF and 30 ng / ml VEGF-C was essentially additive (see FIG. 6). In BAE cells, where both VEGF and VEGF-C individually induce invasion, the response (measured up to 4 days) was much greater than additive as can be seen from the graph of FIG. .

A three-way comparison of the effects of VEGF, VEGF-B and VEGF-C, individually and in combination, is shown in the graphs of FIGS. 8a and 8b. FIG. 8a shows the individual effects of the three cytokines. 30 ng / ml VEGF-
It is understood that the effect produced by B alone is negligible. V
EGF (30 ng / ml) alone produced a significant effect, whereas the greatest single (individual) effect was produced by 30 ng / ml VEGF-C. As can be seen from Figure 8b, when VEGF and VEGF-B were co-administered, the results were clearly greater than the sum of their individual effects. Similarly, VEGF-B and VEGF-
When co-administered with C, the activity of VEGF-C to induce collagen gel penetration was clearly enhanced. However, the most striking effect was that 30 ng / ml VEGF and 30 ng / ml
Produced by combination with ng / ml VEGF-C.

Example 11: PA and PA with VEGF-C in BME and BAE cells
Stimulation of 1-1 activity 15 to VEGF-C at concentrations of 0, 1, 3, 10, 30, and 100 ng / ml.
Zymography (enzyme electrophoresis) and reverse zymography (reverse enzyme electrophoresis) were performed on cell extracts and culture supernatants prepared from BME and BAE cells exposed to time. For comparison purposes, 30 ng / ml VEG each
Test media from BME and BAE cells exposed to F and 10 ng / ml bFGF were also analyzed. Confluent monolayers of endothelial cells in a 35 mm gelatin-coated tissue culture dish were serum-free medium-free.
Washed twice and added each cytokine in serum-free medium containing Trasilol (200 KIU / ml). Fifteen hours later, cell extracts were prepared and analyzed by Vassalli et al., J. Exp. Med. 159: 1653-68 (1984) and pepper (Pep
per) et al., J. Cell Biol., 111: 743-755 (1990) and analyzed by zymography (enzymatic electrophoresis) and reverse zymography (reverse enzymatic electrophoresis).

FIG. 9 a shows zymographic (enzymatic electrophoresis) analysis of cell extracts from BME cells. FIG. 9b shows the same zymography incubated at 37 ° C. for a longer time. FIG. 9c shows a reverse zymograph (reverse enzyme electrophoresis) analysis of the culture supernatant from BAE cells.

Example 12: PA and PA with VEGF-B in BME and BAE cells
Stimulation of I-1 activity The same procedure as in Example 11 was repeated, except that culture supernatant was obtained from cells exposed to VEGF-B at concentrations of 0, 1, 3, 10, 30, and 100 ng / ml. 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 results of zymography (enzymatic electrophoresis) and reverse zymography (reverse enzymatic electrophoresis) shown in FIGS. 9a to 9c show that VEGF-C shows uPA, tPA and PAI- in BME and BAE cells. 1 activity. Similarly, the results of zymography (enzyme electrophoresis) and reverse zymography (reverse enzyme electrophoresis) shown in FIGS.
-B induces uPA and PAI-1 activity in endothelial cells. Induction of uPA and PAI-1 activity was lower than that seen with nearly equimolar VEGF. The results of the zymography test also indicate that bFGF
Confirms that it has little or no effect on tPA expression. The effect of VEGF-C on PAI-1 activity in BME cells, on uPA activity in BAE cells, and on tPA and PAI-1 activity in BLE cells was also tested.
Was not as pronounced as that shown in

Example 13 RNA Preparation, In Vitro Transcription, and Northern Blot Hybridization Cytokines were added to confluent endothelial cell monolayers that had been supplemented 24 hours before with fresh complete medium. Total cell RNA was prepared after the indicated time using Trizol reagent (Gibco BRL, Life Technologies, Paisley, Scotland) according to the manufacturer's instructions. Replicate filters containing total cellular RNA (5 μg / lane) were exposed to VEGF-C or VEGF-B (30 ng / ml) for 0, 1, 3, 9, 24 and 48 hours.
Prepared from a confluent monolayer of cells and hybridized with a ³² P-labeled cRNA probe. Controls for periods of 0, 9, 24 and 48 hours are shown. RNA integrity and homogeneity of the loading were determined by staining the filters with methylene blue after transfer and cross-linking, as shown by the 28S and 18S ribosomal RNAs in the lower figures of FIGS. Northern blots, UV crosslinks, and methylene blue staining of filters, in vitro transcription, hybridization, and post-hybridization washes are described in Pepper et al., J. Cell Biol., 111.
: 743-755 (1990). [ ³² P] -labeled cRNA probe is a bovine urokinase-type plasminogen activator (u-PA) (Kraetzschmar et al., Gene, 125: 177-83 (1993)).
), Bovine u-PA receptor (u-PAR) (Kraetzschmar et al., G
ene, 125: 177-83 (1993)), human tissue-type PA (t-PA) (Fisher
er) 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). VEGF-C was found to increase the steady state levels of uPA, tPA and PAI-1 mRNA in BAE cells.

The Northern blot analysis shown in FIG. 11 shows that VEGF-C
It shows that it increases the steady state levels of PA, uPAR, tPA and PAI-1 mRNA. The kinetics of PA, uPAR and PAI-1 induction were rapid (within 1 hour) and transient (baseline levels were between 3 and 9 hours for uPA and uPAR, with PAI -1
Was achieved between 9 and 24 hours). For uPA and uPAR, this is in contrast to the more sustained 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, this result is similar to that of VEGF and bFGF. The results of the Northern blot test for the test for VEGF-B are shown in FIG.

[0058] Extracellular proteolytic activity, represented by increased uPA synthesis, and PAI-
Co-induction with protease inhibition, represented by an increase in the synthesis of 1, is a feature of angiogenesis both in vivo and in vitro, and is consistent with the theory of "balance of proteolysis" It is something with. The theory is that while proteases are required for cell migration and morphogenesis, protease inhibitors protect the extracellular matrix from inappropriate destruction by
It also recognizes that it plays an important permissive role.

[0059] Example 16: alpha ₂ - by anti-plasmin (antiplasmin), an inhibition BME cell monolayer of endothelial cells angiogenesis in the presence of VEGF-C and bFGF, bFGF and VEGF-C, and 0,1, Co-treated with α ₂ -antiplasmin at concentrations of 3, 10 and 30 μg / ml for 4 days. A single layer below the surface monolayer at a randomly selected area of the processed gel
Photographed at Endothelial cell invasion was quantified as described in Example 5 above. FIG. 13 shows the result in the form of a graph. It can be seen that as the concentration of α ₂ -antiplasmin increases, the angiogenic activity clearly decreases. Thus, it is clear that α ₂ -antiplasmin dose-dependently inhibits the induction of angiogenesis by co-treatment of VEGF-C and bFGF.

The above description and examples are merely illustrative of the invention and are not intended to be limiting. Since modifications of the disclosed embodiments, including the spirit and content of the invention, can be made by one skilled in the art, the invention should be construed to include all within the scope of the claims and their equivalents. It is.

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[Brief description of the drawings]

1a-1f are control micrographs for microphotographs showing cell cord invasion of angiogenesis into collagen gel induced by each factor.

FIG. 1b is a micrograph showing VEGF-induced cell cord invasion of angiogenesis into a collagen gel.

FIG. 1c is a micrograph showing VEGF-C-induced cell cord invasion of angiogenesis into a collagen gel.

FIG. 1d is a micrograph showing cell cord invasion of angiogenesis into a collagen gel induced by bFGF.

1e is a micrograph showing angiogenesis cell cord invasion into a collagen gel induced by a combination of bFGF and VEGF.

FIG. 1f. Micrographs showing angiogenesis cell cord invasion into collagen gels induced by a combination of bFGF and VEGF-C.

FIG. 2a is a graph showing in vitro angiogenesis in bovine microvascular endothelial (BME) cells induced by VEGF-C alone.

FIG. 2b is a graph showing in vitro angiogenesis in bovine microvascular endothelial (BME) cells induced by a combination of VEGF-C and bFGF.

FIG. 3a is a graph showing in vitro angiogenesis in BME cells induced by VEGF-B alone.

FIG. 3b is a graph showing in vitro angiogenesis in BME cells induced by a combination of VEGF-B and bFGF.

FIG. 4a is a graph showing in vitro angiogenesis in bovine aortic endothelial (BAE) cells induced by VEGF-C alone.

FIG. 4b is a graph showing in vitro angiogenesis in bovine aortic endothelial (BAE) cells induced by the combination of VEGF-C and bFGF at a concentration of 1 ng / ml.

FIG. 4c is a graph showing in vitro angiogenesis in bovine aortic endothelial (BAE) cells induced by a combination of VEGF-C and bFGF at a concentration of 10 ng / ml.

FIG. 5a is a graph showing in vitro angiogenesis in BAE cells induced by VEGF-B alone.

FIG. 5b is a graph showing in vitro angiogenesis in BAE cells induced by a combination of VEGF-B and bFGF.

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

FIG. 7: BAE of VEGF and VEGF-C, individually and in combination with each other
Graph showing effect on cell collagen invasion

FIG. 8a is a graph showing the effect of VEGF, VEGF-B and VEGF-C on BAE cell collagen invasion individually.

FIG. 8b is a graph showing the effect of various combinations of VEGF, VEGF-B and VEGF-C on BAE cell collagen invasion.

Figure 9a. Zymograph (enzyme electrophoresis) analysis of the induction of tissue-type plasminogen activator (tPA) activity and urokinase-type plasminogen activator (uPA) activity by VEGF-C, VEGF and bFGF.

Figure 9b. Zymograph (enzyme electrophoresis) analysis of the induction of tissue-type plasminogen activator (tPA) activity and urokinase-type plasminogen activator (uPA) activity by VEGF-C, VEGF and bFGF.

FIG. 9c. Reverse zymograph (reverse enzyme electrophoresis) analysis of the induction of plasminogen activator inhibitor-1 (PAI-1) by VEGF-C, VEGF and bFGF.

Figure 10a: Zymographic analysis of the induction of tPA and uPA activity by VEGF-B, VEGF and bFGF.

FIG. 10b. Reverse zymography (reverse zymography) analysis of the induction of PAI-1 activity by VEGF-B, VEGF and bFGF.

FIG. 11. tPA, uPA and P induced by VEGF-C in BAE cells
Diagram showing elevated steady-state levels of AI-1 mRNA

FIG. 12. uPA and PAI-1 induced by VEGF-B in BAE cells
Diagram showing elevated steady state levels of mRNA

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

──────────────────────────────────────────────────続 き Continuation of the front page (71) Applicant 605 THIRD AVENUE, NEW YORK, NEW YORK 10158, UNITED STATES OF AM ERICA (71) Applicant University of Geneva (Medical Center Faculty of Medicine) Switzerland 1211 Geneva 4 Ryu Michel Selve 1 (72) Inventor Pepper, Michael, Ess Switzerland 1211 Geneva 4 Ryu Michel Selve 1 University of Geneva Medical Center (72) Alitaro, Cali Finland Sef-University of Geneva 00014 University of Helsinki P-O-Box 26 (72) Inventor Ericsson, Ur Sweden es -17,177 Stockholm P. O Box 240 Lou Dovihhi Institute follower A Cancer Research F-term (reference) 4B024 AA01 AA20 BA21 CA04 EA04 FA02 GA11 4B063 QA07 QQ08 QQ43 QR32 QS25 4B065 AA90X AA93X AB01 BB19 BB34 CA44

Claims (25)

[Claims] 1. A method for stimulating angiogenesis in endothelial cells, comprising:
Co-administering a synergistic combination of at least two cytokines selected from the group consisting of F, VEGF-B, VEGF-C and FGF to said cells, wherein said at least two cytokines A method of stimulating angiogenesis in endothelial cells, wherein at least one of the cells is VEGF-B or VEGF-C. 2. The method of claim 1, wherein said endothelial cells are selected from the group consisting of microvascular endothelial cells, aortic endothelial cells, and lymphatic endothelial cells. 3. The method of claim 2, wherein said endothelial cells are bovine endothelial cells. 4. The method of claim 2, wherein said endothelial cells are human endothelial cells. 5. The method, wherein the cells are treated in an in vitro culture in a medium.
2. The method of claim 1, wherein the cytokine is administered by incorporating the cytokine into the medium. 6. The cytokine is administered in the form of a purified protein.
The method of claim 1. 7. An operably linked at least one promoter sequence.
2. The method of claim 1, wherein the cytokine is administered by transfecting or transforming endothelial cells with at least one vector comprising a nucleotide sequence encoding an (operably linked) cytokine. . 8. The method of claim 1, wherein the at least two cytokines are VEGF-C and FGF. 9. The method of claim 1, wherein the at least two cytokines are VEGF-B and FGF. 10. The at least two cytokines are VEGF and VEGF-
The method of claim 1, wherein C is C. 11. The method of claim 11, wherein the at least two cytokines are VEGF-B and VEG.
2. The method of claim 1, wherein the method is FC. 12. The at least two cytokines are VEGF and VEGF-
The method of claim 1, wherein B is B. 13. An endothelial cell angiogenesis by cells which release a synergistic combination of at least two angiogenic cytokines selected from the group consisting of VEGF, VEGF-B, VEGF-C and FGF. A method of modulating, wherein at least one of said at least two cytokines is VEGF-B or VEGF-C, wherein said method neutralizes one of said at least two angiogenic cytokines To do (n
eutralizing), which modulates endothelial cell angiogenesis. 14. The method of claim 13, wherein the neutralizing is performed by treating the cells with an antibody such that angiogenic cytokines are neutralized.
The method described in. 15. The method according to claim 1, wherein the cytokine to be neutralized is VEGF-C.
4. The method according to 4. 16. The method according to claim 1, wherein the cytokine to be neutralized is VEGF-B.
4. The method according to 4. 17. The method according to claim 14, wherein said antibody is a monoclonal antibody. 18. The method of claim 13, wherein said cells are solid tumor cells. 19. A method for inhibiting angiogenesis by endothelial cells in the presence of VEGF-C and FGF, comprising treating the cells with anti-plasmin. 20. The method according to claim 19, wherein said antiplasmin is α ₂ -antiplasmin. 21. 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. 22. A method for stimulating the proteolytic activity of endothelial cells, wherein the cells are VEGF-B, VEGF-C or a combination of VEGF-B and VEGF-C, and at least one selected from VEGF and FGF. A method for stimulating the proteolytic activity of endothelial cells, comprising the step of treating with one additional cytokine. 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. 24. A method of inhibiting endothelial cell penetration, invasion and / or metastasis in a patient, comprising administering to said patient an amount of VEG effective to inhibit endothelial cell proliferation.
A method of inhibiting endothelial cell penetration, invasion and / or metastasis in a patient, comprising administering an antagonist of FB or VEGF-C. 25. A method for regulating angiogenic activity of endothelial cells, comprising transfecting or transforming cells with a vector containing an antisense nucleic acid against VEGF-B or VEGF-C. A method of regulating the angiogenic activity of a cell.