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WO1994005777A1 - Inhibition de la formation ou de l'activite du processus de 12-lipoxygenase dans les leucocytes humains - Google Patents

Inhibition de la formation ou de l'activite du processus de 12-lipoxygenase dans les leucocytes humains Download PDF

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WO1994005777A1
WO1994005777A1 PCT/US1993/008106 US9308106W WO9405777A1 WO 1994005777 A1 WO1994005777 A1 WO 1994005777A1 US 9308106 W US9308106 W US 9308106W WO 9405777 A1 WO9405777 A1 WO 9405777A1
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human
cells
rna
expression
glucose
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PCT/US1993/008106
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Jerry L. Nadler
Rama Devi Natarajan
Jiali Gu
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City Of Hope
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Priority to AU48403/93A priority Critical patent/AU674339B2/en
Priority to JP6507341A priority patent/JPH07500254A/ja
Priority to EP93921232A priority patent/EP0621895A4/en
Publication of WO1994005777A1 publication Critical patent/WO1994005777A1/fr

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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
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    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C12Y113/11012—Linoleate 13S-lipoxygenase (1.13.11.12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C12Y113/11031—Arachidonate 12-lipoxygenase (1.13.11.31), i.e. lipoxygenase-type-12
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • This invention relates to a new form of 12-lipoxygenase (12-LO) RNA and protein in human adrenal, vascular smooth muscle, endothelial and mononuclear cells.
  • the invention also relates to the mediation of angiotensin II (All) and glucose induced vascular and renal actions by activation of this 12-LO pathway.
  • Enhanced atherosclerotic cardiovascular and renal disease continue to be major causes of morbidity and mortality in patients with diabetes ellitus and hypertension. All activity and elevated glucose are known to play a role in increased propensity to these disorders.
  • the 12-LO pathway can produce active products including 12-hydroperoxyeicosatetra- enoic acid (12-HPETE) and more stable 12 hydroxyeico- satetraenoic acid (12-HETE) .
  • some 12-LO enzymes can also metabolize linoleic acid to produce additional active lipids called hydroxyoctadecadienoic acid (HODES) .
  • HODES hydroxyoctadecadienoic acid
  • the LO products may play a key role in the development of vascular and renal disease.
  • 12-HETE and 12-HPETE have been shown to be important mediators of All induced effects on inhibition of renin release from kidney (1) stimulation of aldosterone synthesis from rat and human adrenal cells (2,3) and increase in blood pressure in rats (4) .
  • 12-HETE and 12-HPETE can lead to vascular smooth muscle cell migration at concentrations as low as 10-14M (5) , and both products can inhibit the synthesis of the vasoprotective eicosanoid prostacyclin (6,7) .
  • the linoleic acid metabolities including 13 and 9 HODE have been recently found to be capable of producing mitogenic effects in certain cell types including the liver and fibroblasts (8,9) and can mediate epidermal growth factor induced proliferative actions (9) .
  • Recent studies in several species have shown the presence of two forms of 12-LO (10,11).
  • One type has been cloned from porcine leukocytes (10) , which shares 85% sequence homology to a human tracheal 15-LO enzyme (12) .
  • Another type of 12-LO found almost exclusively in human platelets is only 65% homologous to the porcine leukocyte type of 12-LO (11) .
  • These two forms of 12-LO not only differ in amino acid sequence, but also show differences in preferred substrates.
  • the platelet type of 12-LO exclusively reacts with arachiodonic acid to form 12-HETE.
  • the porcine leukocyte type of 12-LO reacts with linoleic acid and to form 9 and 13-HODE as well as arachidonic acid to form 12-HETE.
  • 12-LO products can mediate All-induced bovine adrenal cell proliferation (13) .
  • This invention includes the discovery of a new form of 12-LO RNA and protein in human adrenal, mononuclear, vascular smooth muscle and endothelial cells. Activation of this 12-LO pathway plays a key role in mediating All and glucose induced vascular and renal actions. Products of this newly discovered 12-LO pathway can directly activate protein kinase C and lead to increased vascular smooth muscle cell growth, a hallmark atherosclerotic vascular disease.
  • Another aspect of the invention postulates treatment or prevention of vascular disease in patients with diabetes mellitus or hypertension by inhibition of this new form of 12-LO.
  • the invention thus provides a rationale for development of a new pharmaceutical or molecular method to inhibit this newly discovered lipoxygenase pathway.
  • Figure 1 is a bar graph which depicts the effect of All on 12- and 15-HETE released by porcine smooth muscle cells (SMC) grown in normal glucose.
  • Figure 2 is a bar graph which depicts the effect of All on cell associated 12-HETE levels in porcine vascular smooth muscle cells (PVSMC) cultured in normal (5.5 mM) or high (25 mM) glucose.
  • PVSMC porcine vascular smooth muscle cells
  • Figure 3 is a bar graph which depicts the effect of high glucose (25 mM) on 12- and 15-HETE levels in porcine aortic smooth muscle cells.
  • Figure 4 is a Western immunoblot which depicts the effect of All (10 ⁇ 7 M) on 12-LO 72K) expression in PVSMC in normal (5.5mM) or high (25mM) glucose.
  • Figure 5 is a Western immunoblot which depicts specific porcine leukocyte 12-LO protein expression in PVSMC. Lane 1, antigen-porcine 12-LO; Lanes 2 and 3, PVSMC cytosols. A: With 12-LO antibody 1:600, B: With 12-LO antibody preincubated with 12-LO antigen.
  • FIG. 6 is a Southern blot analysis of 12-LO mRNA levels in PVSMC by reverse transcriptase PCR (RT-PCR) .
  • RT-PCR reverse transcriptase PCR
  • Figure 7 depicts a Southern blot analysis which shows regulation of 12-LO mRNA by All in PVSMC.
  • Figure 8 is a bar graph which depicts the effect of All on protein synthesis in PVSMC cultures in normal or high glucose.
  • Figure 9 is a bar graph which depicts the effect of baicalein, a 12-LO synthesis inhibitor, on All-induced DNA and protein synthesis in PVSMC.
  • Figure 10 is a bar graph which depicts protein synthesis after direct addition of 12 and 15-LO products in PVSMC cultured in normal glucose.
  • Figure 11 is a bar graph which depicts protein synthesis after addition of 12 and 15-LO products in PVSMC cultured in high glucose.
  • Figure 12 depicts growth curves of PVSMC in normal and high glucose.
  • Figure 13 illustrates the regulation of 12-LO protein expression by All in human adrenal glomerulosa cells.
  • Figure 14 is a Southern blot analysis showing expression of 12-LO RNA in human adrenal glomerulosa and U937 mononuclear cells.
  • Figure 15 is a Northern blot analysis that shows expression of 4.1 kb size 12-LO RNA band in human adrenal glomerulosa.
  • Figure 16 is a Southern blot analysis that illustrates the regulation of 12-LO RNA levels by All as determined by RT-PCR.
  • Figure 17 depicts regulation of 12-LO protein expression by All in human aortic vascular smooth muscle (HVSMC) .
  • Figure 18 illustrates the presence of leukocyte type 12-LO in human aortic smooth muscle and mononuclear cells and also shows induction of 12-LO expression by All in human vascular smooth muscle cells (HSMC) .
  • HSMC human vascular smooth muscle cells
  • Figure 19 shows the release of 12-LO product 12-HETE by All in human vascular smooth muscle cells.
  • Figure 20 depicts the effect of baicalein (10 ⁇ 6 M) a 12-LO inhibitor on smooth muscle cell growth in normal glucose (5.5 mM) and high glucose (25 mM) conditions. Cell number is significantly reduced by baicalein in high glucose only. v High glucose without baicalein
  • Figure 21 depicts RT-PCR Southern blot analysis showing the presence of human leukocyte type 12-LO in human aortic endothelial cells.
  • Lane 1 cDNA positive control.
  • Lane 2 12-LO expressed DNAase treated showing band is not from DNA contamination and
  • Lane 3 is total RNA from endothelial cells showing 333 base pair product.
  • Figure 22 depicts RT-PCR, Southern blot analysis showing that human aortic endothelial cells do not express the 15-LO RNA but only the 12-LO RNA of leukocyte type. Presence of positive control amplification of 15-LO cDNA but complete absence of 15-LO RNA in two separate samples of human aortic endothelial cells is depicted.
  • Figure 23 shows specificity of PCR method for amplification and expression of either leukocyte 12-LO or 15-LO RNA.
  • Figure 4 is a Western immunoblot using an antibody against porcine leukocyte 12-LO showing effects of high glucose (25mM) and All (10 ⁇ 7 M) on 12-LO enzyme (72 KD) expression at 45 hours. It is clearly seen that basal 12-LO enzyme expression is markedly increased in PVSMC cultured in high glucose. In addition, All caused a significant stimulation in 12-LO expression in normal ad high glucose. The specificity of these results using antibody blocking studies was also confirmed.
  • Figure 5 shows that the bands obtained with authentic porcine leukocyte 12-LO enzyme (lane 1) as well as with PVSMC cytosols (lanes 2 and 3) (A) all disappeared when treated with 12-LO antibody which had been preincubated for two hours with the 12-LO enzyme (B) .
  • PVSMC express the leukocyte form of 12-LO and that porcine 12-LO enzyme expression is increased by high glucose as well as All.
  • the invention also includes the discovery that All, as well as high glucose, can upregulate 12-LO mRNA expression.
  • a specific reverse transcriptase polymerase chain PCR procedure was designed for evaluating basal and stimulated 12- and 15-LO mRNA levels in PVSMC, human adrenal glomerulosa, human vascular smooth muscle and monocytes.
  • the sequences of the primers and the probes were designed based on known gene sequences (10,12,13,14) , and selected from regions displaying most divergence between porcine 12-LO and 15-LO sequences (11) .
  • SEQ ID. 1 Primer 1:5'AACTCAAGGTGGAAGTACCGGAG3' nucleotides 146 to 168
  • SEQ ID. 2 Primer 2:5 , ATATAGTTTGGCCCCAGCCATATTC3' complementary to nucleotides 453 to 477
  • SEQ ID. 3 Probe: 5'AGGCTCAGGACGCCGTTGCCC3 ' complementary to nucleotides 306 to 326.
  • Porcine Leukocyte 12-LO (Ref. No. 10) .
  • SEQ ID. 4 Primer 1:5' TTCAGTGTAGACGTGTCGGAG3' nucleotides 145 to 165.
  • SEQ ID. 5 Primer 2:5' ATGTATGCCGGTGCTGGCTATA TTTAG 3' complementary to nucleotides 451 to 477.
  • SEQ ID. 6 Probe: 5' TCAGGATGCGGTCGCCCTCCAC 3' complementary to nucleotides 301 to 322.
  • RNA from both human adrenal glomerulosa tissue and cultured cells was extracted with guanidium thiocyanate-phenol-chloroform using RNAzol (Cinna/Biotecx Laboratories International, Inc., Texas) .
  • Poly (A) + RNA was purified by oligo (dT) cellulose chromatography column (5 prime > 3
  • RNA or mRNA was mixed with the PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.001% gelatin) , 200 ⁇ M of each of the four deoxynucleotide trisphosphates, 25 pmole each of 5' and 3' primers 5'TTCAGTGTAGACGTGTCGGAG3' (SEQ ID. 4) and 5'ATGTATGCCGGTGCTGGCTATATTTAG3' (SEQ ID. 5), 2 units of Avian Myeloblastosis Virus reverse transcriptase (20 U/ul, Lie Sciences, St.
  • RNA samples from HEL cells or IM-9 cells were run as controls in both PCR nd in Northern analysis.
  • the human 15-LO cDNA, porcine leukocyte 12-LO cDNA and human platelet 12-LO cDNA amplifications were carried out by mixing 2-5 ng of cDNA in 50 ⁇ l volume containing 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl , 0.001% gelatin, 200 ⁇ M of each of the four deoxynucleotide triphosphates, 25 pmole of 5' and 3 ' primers, and 2.5 U of Taq polymerase.
  • the size of the amplified fragment is 333 bp for both of 12-LO and 15-LO.
  • the 333 bp PCR amplified fragment obtained with porcine leukocyte 12-LO cDNA or with human 15-LO cDNA as a template could be seen in an ethidium bromide stained gel after 25 cycles of amplification (data not shown) .
  • the 333 bp amplified product could not be seen in an ethidium bromide stained gel even after 35 cycles of amplification.
  • the product could only be detected by autoradiography of a blot hybridized with a porcine leukocyte 12-LO oligonucleotide probe.
  • porcine leukocyte 12-LO cDNA probe could not readily distinguish the 333 bp amplified products corresponding to porcine leukocyte 12-LO or to human 15-LO (data not shown) .
  • human 15-LO oligonucleotide and porcine leukocyte 12-LO oligonucleotide probes can cross hybridize to the 12-LO or 15-LO amplified product, respectively using 12-LO or 15-LO cDNA as templates of amplification.
  • high stringency e.g., hybridized membrane wash temperature of 60°C
  • the 333 bp PCR amplified products of porcine 12-LO and human 15-LO were distinguished by the cDNA probe.
  • Figure 23 depicts comparison autoradiograms of PCR of cDNA for human 15-LO and cDNA for porcine leukocyte 12-LO.
  • cDNAs samples were amplified for 25 cycles with specific primers for the gene (Table 1) and were hybridized with a labeled porcine leukocyte 12-LO oligonucleotide probe (panel A) or with a labeled human 15-LO oligonucleotide probe (panel B) .
  • Lane 1 is porcine leukocyte 12-LO primers on cDNA for porcine leukocyte 12-LO.
  • Lane 2 is human 15-LO primers on cDNA for human 15-LO.
  • ATGTATGCCGGTGCTGGCTATATTTAG3' (SEQ ID. 5)
  • dNTP 2 ⁇ g of RNA in a final volume of 9 ⁇ l.
  • the mixture was heated to 80°C for 5 minutes and cooled to 37°C.
  • Two units of AMV reverse transcriptase was added and maintained for three minutes at 37°C.
  • an additional two units of AMV reverse transcriptase was added, the sample was heated to 95°C to denature, and then amplified for 40 cycles by PCR as described before.
  • the PCR product was analyzed by hybridization.
  • Sequencing reaction of purified PCR product of U937 cells was set in the presence of 0.5% NP-40 detergent and [ 7 32 -P] ATP labeled porcine leukocyte 12-LO oligonucleotides 145-165, 451-477 and 301-322.
  • DNA sequencing reactions were performed by the dideoxynucleotide chain termination method using Sequenase (United States Biochemicals, Cleveland, Ohio) , sequencing in both directions with 5' primer and 3 primers.
  • FIG. 6 is a Southern blot analysis of the RT-PCR (25 cycles) amplified products from PVSMC total RNA. Hybridization was performed with the porcine leukocyte type 32 P-labeled 12-LO oligonucleotide probe. It is seen that cells cultured in high glucose have a much greater expression of the 333 bp 12-LO PCR amplified product than those cultured in normal glucose. GAPDH mRNA amplification was used as an internal standard (280 bp) .
  • Figure 8 shows that All (10"-%) increased total cell protein (126% of control) in PVSMC cultured in normal glucose. Similar results were obtained with All 10 ⁇ 7 and 10 ⁇ 8 M. However, the effects of All on total cell protein were significantly greater in PVSMC grown in elevated glucose (147% of control) . These results indicate that elevated glucose enhances the hypertrophic response of All.
  • the hypertrophic response of All is mediated at least in part by activation of the 12-LO pathway.
  • the role of the LO pathway in All-induced hypertrophic effects is illustrated by Figure 9 which shows that All-induced protein synthesis in normal glucose was blunted by a specific 12-LO inhibitor baicalein. Similar results were obtained in high glucose.
  • Figure 10 shows that the 12-LO product 12-HETE could directly increase protein synthesis with the same potency as All in normal glucose.
  • the effect of not only All, but also 12-HETE was enhanced in elevated glucose (Figure 11) .
  • 15-HETE was less potent than 12-HETE showing significant effects only in elevated glucose ( Figures 10 and 11) .
  • Table 1 shows the results of Protein Kinase C (PKC) activity measurements in PVSMC grown in normal and high glucose. TABLE 1
  • PKC isoforms may be a key mechanism for vascular cell proliferation in response to glucose All and the LO products.
  • FIG. 13 shows the effect of All (10 ⁇ 7 M) on the expression of the 12-LO protein in normal human adrenal glomerulosa cells as assessed by Western immunoblotting. All increased the expression of 12-LO (Fig. 13A) approximately two-fold over basal as determined by densitometric analysis (Fig. 13B) .
  • the 12-LO protein is present in cultured human glomerulosa cells as seen using an antibody against a porcine leukocyte 12-LO.
  • the 12-LO protein expression is increased in cells cultured in the presence of All for 30 hours.
  • RNA samples were amplified for 30 cycles with SEQ ID. 4 and 5 porcine leukocyte 12-LO primers.
  • Membranes were hybridized with internal porcine leukocyte 12-LO oligonucleotide probe (SEQ ID. 6) .
  • Panel A, lane 1 represents total RNA from normal human adrenal glomerulosa using RT-PCR.
  • Lane 2 is a negative control without template and lane 3 is a negative control using human 15-LO cDNA.
  • Samples in panel B are mRNA or total RNA from human U937 cells. Lanes 1 and 5 represent negative controls without reverse transcriptase (RT) for mRNA and total RNA respectively. Lane 2, mRNA and lane 6, total RNA are true RT-PCR. Lane 3 is a positive control using the porcine leukocyte 12-LO cDNA. Lane 4 is another negative control without RNA template.
  • RT reverse transcriptase
  • Figure 15 is a Northern analysis using the 12-LO probe (SEQ ID. 6) . 20 ug of RNA from human adrenal glomerulosa in lanes 1 and 2 showing that the size of the RNA expressed (approximately 4.1 kb) is similar to the porcine leukocyte 12-LO RNA size.
  • Figure 16 shows regulation of 12-LO mRNA levels by All determined by RT-PCR.
  • Total RNA was extracted from cultured adrenal glomerulosa cells that were incubated alone or with 10 ⁇ 7 M All for 24 hours.
  • RNA samples were amplified for 25 cycles with primers amplifying porcine 12-LO. All reactions in the experiment also contained primers amplifying human GAPDH. Controls without RNA or with RNA pretreated with RNAase were simultaneously run. The position of the specific products are indicated by arrows.
  • 284 bp and 333 bp represent amplified products of human GAPDH and porcine leukocyte 12-LO respectively.
  • Panel A is the autoradiogram of the blot hybridized with oligonucleotide probe specific for the porcine 12-LO gene.
  • Panel B is the autoradiogram of the same blot subsequently hybridized with oligonucleotide probe for the GAPDH.
  • Lanes 1 and 4 are glomerulosa cells in the control incubation.
  • Lanes 2 and 5 are glomerulosa cells incubated with 10 ⁇ 7 M All. Samples in lanes 4 and 5 were treated with RNase A prior to the RT-PCR.
  • Lane 3 is without RNA.
  • the amplified PCR product in human monocytes and adrenal cells was not due to contamination of the porcine 12-LO cDNA.
  • the amplified product was sequenced. As shown in Table 2, the sequence in human cells is 2 base pairs different than the porcine sequence.
  • amplified genomic DNA from human leukocyte nuclei shows that the gene size in the segment amplified (1 Kb) was substantially larger than the expected size in the same region in the porcine gene.
  • Figure 17 shows identical procedures as outlined previously for protein expression that All can increase 12-LO protein expression in human aortic smooth muscle cells. The increase of expression was seven fold as measured using a computerized video densitometric system.
  • Figure 18 shows expression of 12-LO RNA in human vascular smooth muscle cells using a similar RT-PCR procedure. Lane 5 shows expression of the expected 333 base pair 12-LO band in human vascular smooth muscle cells, while lane 3 shows an identical RNA band in samples taken from mononuclear cells. Basal expression of 12-LO in unstimulated smooth muscle cells is below the detection limit of this experiment (lane 7) . However, smooth muscle cells stimulated by All show a marked increase in 12-LO expression (lane 5) .
  • Panel B represents an ethidium bromide stain of the RT-PCR experiment showing internal marker RNA (B 2 micoglobulin) for these experiments (lanes 2, 4, 6) .
  • Figure 19 illustrates the stimulatory effect of All at 10 ⁇ 9 and 10 ⁇ 8 M on 12-HETE synthesis and release from human aortic smooth muscle cells. 12-HETE was assayed by HPLC and specific radioim unoassay.
  • FIG. 21 depicts RT-PCR Southern blot analysis showing the presence of human leukocyte type 12-LO in human aortic endothelial cells. Lane 1, cDNA positive control. Lane 2, total RNA from endothelial cells that have been treated by DNAase showing that band is not from DNA contamination and Lane 3 is total RNA from endothelial cells showing 333 base pair product. These results suggest that a 12-LO is expressed in this key vascular wall.
  • Figure 22 depicts the evidence against a 15-LO being expressed in human aortic endothelial cells.
  • 15-LO specific primers and probes revealed specific amplification of the 15-LO cDNA used as a template.
  • 15-LO RNA band is seen when RNA from endothelial cells is used. Therefore, only a leukocyte type of 12-LO is expressed in human aortic endothelial cells.
  • 12-LO gene The presence of a new form of 12-LO gene in human tissues has been described. This 12-LO gene appears to encode a protein which forms active products that mediate angiotensin II and glucose-induced vascular and probably renal actions.

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  • Food Science & Technology (AREA)
  • Cardiology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne la découverte d'une nouvelle forme d'ARN de 12-lipoxygénase et d'une protéine dans les muscles lisses vasculaires, mononucléés, surrénaux et dans les cellules endothéliales de l'homme. L'activation de ce processus 12-LO régule l'angiotensine II et les activités rénales et vasculaires induites par le glucose. L'invention justifie le développement des procédés pharmaceutiques ou moléculaires permettant d'inhiber le processus de la lipoxygénase récemment découvert.
PCT/US1993/008106 1992-08-28 1993-08-26 Inhibition de la formation ou de l'activite du processus de 12-lipoxygenase dans les leucocytes humains WO1994005777A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU48403/93A AU674339B2 (en) 1992-08-28 1993-08-26 Inhibition of the formation or activity of human leukocyte 12-lipoxygenase pathway
JP6507341A JPH07500254A (ja) 1992-08-28 1993-08-26 ヒト白血球１２−リポキシゲナーゼ経路の形成または活性の阻害
EP93921232A EP0621895A4 (en) 1992-08-28 1993-08-26 Inhibition of the formation or activity of human leukocyte 12-lipoxygenase pathway.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US93666092A 1992-08-28 1992-08-28
US07/936,660 1992-08-28

Publications (1)

Publication Number Publication Date
WO1994005777A1 true WO1994005777A1 (fr) 1994-03-17

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Family Applications (2)

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PCT/US1993/008106 WO1994005777A1 (fr) 1992-08-28 1993-08-26 Inhibition de la formation ou de l'activite du processus de 12-lipoxygenase dans les leucocytes humains
PCT/US1994/000089 WO1995018609A1 (fr) 1992-08-28 1994-01-04 12-lipoxygenase de leucocytes humains, mediation exercee par la voie metabolique de cette enzyme et consequences de cette action

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US1994/000089 WO1995018609A1 (fr) 1992-08-28 1994-01-04 12-lipoxygenase de leucocytes humains, mediation exercee par la voie metabolique de cette enzyme et consequences de cette action

Country Status (5)

Country Link
EP (2) EP0621895A4 (fr)
JP (1) JPH07500254A (fr)
AU (2) AU674339B2 (fr)
CA (1) CA2077461C (fr)
WO (2) WO1994005777A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034943A1 (fr) * 1995-05-04 1996-11-07 City Of Hope 12-lipoxygenase leucocytaire humaine et son role dans la pathogenese d'etats pathologiques
US6103496A (en) * 1998-05-29 2000-08-15 Vanderbilt University Isolated and purified 12R-lipoxygenase protein and nucleic acids
WO2000061765A3 (fr) * 1999-04-12 2001-02-22 Lexicon Genetics Inc Nouvelles proteines de lipoxygenase et polynucleotides codant pour celles-ci
WO2001048167A1 (fr) * 1999-12-27 2001-07-05 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, lipoxygenase 10, et polynucleotide codant pour ce polypeptide
WO2001068823A1 (fr) * 2000-03-15 2001-09-20 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, lipoxydase humaine 9, et polynucleotide codant pour ce polypeptide
US6893829B2 (en) 1992-08-28 2005-05-17 City Of Hope Human leukocyte 12-lipoxygenase and its role in the pathogenesis of disease states

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPP349098A0 (en) * 1998-05-13 1998-06-04 South Eastern Sydney Area Health Service A method of monitoring pancreatic tissue viability

Citations (1)

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US4849445A (en) * 1983-12-14 1989-07-18 The Upjohn Company Method for treating or preventing deep vein thrombosis using lipoxygenase inhibitors

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JPH0815851B2 (ja) * 1987-10-09 1996-02-21 日産自動車株式会社 差動制限装置

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US4849445A (en) * 1983-12-14 1989-07-18 The Upjohn Company Method for treating or preventing deep vein thrombosis using lipoxygenase inhibitors

Non-Patent Citations (9)

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Title
European Journal of Pharmacology, Volume 172, Number 3, issued 15 August 1989, A.J. ROBINSON-WHITE et al., "Inhibition of inositol phospholipid hydrolysis in endothelial cells by pentobarbital", pages 291-303, especially the Abstract. *
FASEB Journal, Volume 6, issued February 1992, J. GU et al., "Evidence for expression of a new form of 12-lipoxygenase (12-LO) in human cells", page A1564, Abstract No. 3638, see the entire document. *
Journal of Biological Chemistry, Volume 266, Number 19, issued 05 July 1991, C.D. FUNK et al., "Eicosanoid Forming Enzyme mRNA in Human Tissues: Analysis by Quantitative Polymerase Chain Reaction", pages 12508-12513. *
Proceedings of the National Academy of Sciences of the USA, Volume 87, issued August 1990, C.D. FUNK et al., "Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase", pages 5638-5642. *
Proceedings of the National Academy of Sciences of the USA, Volume 87, issued March 1990, T. YOSHIMOTO et al., "Cloning and sequence analysis of the cDNA for arachidonate 12-lipoxygenase of porcine leukocytes", pages 2142-2146, especially pages 2144-2145. *
Proceedings of the National Academy of Sciences of the USA, Volume 87, issued October 1990, T. IZUMI et al., "Cloning of the cDNA for human 12-lipoxygenase", pages 7477-7481. *
Proceedings of the National Academy of Sciences of the USA, Volume 89, issued May 1992, C.D. FUNK et al., "Characterization of human 12-lipoxygenase genes", pages 3962-3966. *
Proceedings of the National Academy of Sciences of the USA, Volume 90, Number 11, issued June 1993, R. NATARAJAN et al., "Elevated glucose and angiotensin II increase 12-lipoxygenase activity and expression in porcine aortic smooth muscle cells", pages 4947-4951. *
See also references of EP0621895A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6893829B2 (en) 1992-08-28 2005-05-17 City Of Hope Human leukocyte 12-lipoxygenase and its role in the pathogenesis of disease states
WO1996034943A1 (fr) * 1995-05-04 1996-11-07 City Of Hope 12-lipoxygenase leucocytaire humaine et son role dans la pathogenese d'etats pathologiques
US6103496A (en) * 1998-05-29 2000-08-15 Vanderbilt University Isolated and purified 12R-lipoxygenase protein and nucleic acids
US6569644B2 (en) 1998-05-29 2003-05-27 Vanderbilt University Isolated and purified 12R-lipoxygenase protein and nucleic acids
WO2000061765A3 (fr) * 1999-04-12 2001-02-22 Lexicon Genetics Inc Nouvelles proteines de lipoxygenase et polynucleotides codant pour celles-ci
US6582957B1 (en) 1999-04-12 2003-06-24 Lexicon Genetics Incorporated Lipoxygenase proteins and polynucleotides encoding the same
US7144730B2 (en) 1999-04-12 2006-12-05 Lexicon Genetics Incorporated Lipoxygenase proteins and polynucleotides encoding the same
WO2001048167A1 (fr) * 1999-12-27 2001-07-05 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, lipoxygenase 10, et polynucleotide codant pour ce polypeptide
WO2001068823A1 (fr) * 2000-03-15 2001-09-20 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, lipoxydase humaine 9, et polynucleotide codant pour ce polypeptide

Also Published As

Publication number Publication date
AU674339B2 (en) 1996-12-19
JPH07500254A (ja) 1995-01-12
WO1995018609A1 (fr) 1995-07-13
EP0696193A4 (fr) 1996-11-27
EP0696193A1 (fr) 1996-02-14
EP0621895A1 (fr) 1994-11-02
CA2077461C (fr) 2000-02-15
AU4840393A (en) 1994-03-29
EP0621895A4 (en) 1996-11-27
AU7090694A (en) 1995-08-01
CA2077461A1 (fr) 1994-03-01

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