WO1994005777A1

WO1994005777A1 - Inhibition of the formation or activity of human leukocyte 12-lipoxygenase pathway

Info

Publication number: WO1994005777A1
Application number: PCT/US1993/008106
Authority: WO
Inventors: Jerry L. Nadler; Rama Devi Natarajan; Jiali Gu
Original assignee: City Of Hope
Priority date: 1992-08-28
Filing date: 1993-08-26
Publication date: 1994-03-17
Also published as: AU4840393A; EP0696193A4; WO1995018609A1; EP0621895A1; AU7090694A; CA2077461A1; JPH07500254A; CA2077461C; EP0696193A1; EP0621895A4; AU674339B2

Abstract

The discovery of a new form of 12-lipoxygenase RNA and protein in human adrenal, mononuclear, vascular smooth muscle and endothelial cells is disclosed. Activation of this 12-LO pathway mediates angiotensin II and glucose induced vascular and renal actions. A rationale for the development of pharmaceutical or molecular methods to inhibit this newly discovered lipoxygenase pathway is described.

Description

INHIBITION OF THE FORMATION OR ACTIVITY OF HUMAN LEUKOCYTE 12-LIPOXYGENASE PATHWAY

This application is a continuation of United States application, Serial No. 07/936,660, filed 28 August 1992.

This invention was made with government support under Grant No. DK 39721 ROl awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION

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.

Applicant has found that All can increase the activity of a 12-lipoxygenase (12-LO) pathway of arachidonic acid. The 12-LO pathway can produce active products including 12-hydroperoxyeicosatetra- enoic acid (12-HPETE) and more stable 12 hydroxyeico- satetraenoic acid (12-HETE) . In addition, some 12-LO enzymes can also metabolize linoleic acid to produce additional active lipids called hydroxyoctadecadienoic acid (HODES) .

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) . Furthermore, 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. However, 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. In a recent study it has also been found that 12-LO products can mediate All-induced bovine adrenal cell proliferation (13) .

Prior to this invention it was not known that a leukocyte type of 12-LO is also expressed in human tissue. SUMMARY OF THE INVENTION

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.

DESCRIPTION OF THE FIGURES

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.

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 (10~⁷M) 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.

Figure 6 is a Southern blot analysis of 12-LO mRNA levels in PVSMC by reverse transcriptase PCR (RT-PCR) . The results show marked increase in 12-LO expression in cells cultured in high glucose. There was no effect of glucose on GAPDH (marker gene) expression.

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) .

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 (10~⁶M) 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

T High glucose with baicalein o Normal glucose without baicalein

• Normal glucose with 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.

DETAILED DESCRIPTION OF THE INVENTION

I. Profile and Mechanism of LO Product Formation

LO product formation: In PVSMC cells cultured in normal glucose (5.5 mM, 100 mg/dl), All increased the release of both 12- and 15-HETE into the media (Figure 1) . However, All did not increase cell associated HETE levels in normal glucose (Figure 2) . In contrast, cells grown in high glucose (25 mM, 450 mg/dl) showed markedly elevated levels of cell associated 12- as well as 15-HETE (Figure 3) (12-HETE; 1413±174 normal glucose vs 2600±181 high) and 15-HETE: (15301134 normal vs 33221225 high pg/10⁶ cells, both p<0.01 high vs normal glucose). Figure 2 also shows that All further increased the cell associated 12-HETE concentrations in high glucose.

These results indicate that All stimulates 12- and 15-LO product (HETE) formation in PVSMC. In addition, PVSMC cultured in elevated glucose media produce more LO products and have an enhanced LO response to All.

LO protein expression: Figure 4 is a Western immunoblot using an antibody against porcine leukocyte 12-LO showing effects of high glucose (25mM) and All (10~⁷M) 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) .

These results indicate that PVSMC express the leukocyte form of 12-LO and that porcine 12-LO enzyme expression is increased by high glucose as well as All.

LO mRNA expression: The invention also includes the discovery that All, as well as high glucose, can upregulate 12-LO mRNA expression. To demonstrate this discovery, 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) .

Human 15-LO (Ref. 12) . All sequences are 5 ' -3 - .

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.

Total 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

Prime, Inc. , West Chester Pennsylvania) . 1 μg of total RNA or mRNA was mixed with the PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl₂, 0.001% gelatin) , 200 μM 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. Petersburg, FL) and 2.5 units Taq polymerase (Perkin Elmer Cetus) , in a final volume of 50 μl . In some reactions, 5 pmole of each 5' and 3' primers of β2 microglobuiline or GAPDH were added as an internal standard. The samples were placed in a thermal cycler at 37°C for 8 minutes for the reverse transcriptase reaction to proceed. Then conditions used for PCR were a denaturation step at 94°C for 1 minute, annealing at 50°C for 2 minutes and extension at 72°C for 2 minutes for 25-30 cycles. Blank reactions with no RNA template, or with no reverse transcriptase were carried out through the RT nd PCR steps. 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 μl volume containing 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl , 0.001% gelatin, 200 μM 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) . However, using RNA samples from the human cells, 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.

Since the amino acid sequences of porcine leukocyte 12-LO and human 15-LO are highly homologous, the 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) . Moreover, 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. At 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.

Direct DNA sequencing of PCR product

PCR amplification: RNA was reverse transcribed as follows: the reaction mixture contained porcine leukocyte 12-LO complementary primer

5^,ATGTATGCCGGTGCTGGCTATATTTAG3' (SEQ ID. 5), dNTP and 2 μg of RNA in a final volume of 9 μl. 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. Then 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. In order to obtain sufficient amount of PCR product for sequencing, 1 μl of total product of the PCR reaction was used as a template for secondary PCR amplification with 5' and 3' primers, (145-165 and 451-477) primers for U937 cells; (177-198 and 428-450) primers for human adrenal. The reaction conditions were as described before except that 30 cycles was used.

Preparation of DNA for sequencing: The products of secondary PCR were purified by electrophoresis on nondenaturing 8% polyacrylamide gel. The isolated DNA fragment was directly used for sequencing.

Sequencing: Two approaches were used for sequencing. (A) 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.

(B) Sequencing reaction of PCR products of human adrenal glomerulosa tissue and U937 cells was performed by a cycle sequencing method of AmpliTaq DNA polymerase with a cycle sequence kit (Perkin Elmer Cetus, Norwalk, CT) . 15 ng of human adrenal PCR product and 2 pmole of [₇ ³²-P] ATP labeled porcine leukocyte 12-LO oligonucleotide (177-198 or 428-450) were used. The cycling program was 1 minute at 95°C, and 1 minute at 60°C for 20 cycles.

As Figure 23 shows, amplification of their cDNAs has confirmed that specific expression of leukocyte type 12-LO and human 15-LO is accomplished.

Regulation of Porcine 12-LO mRNA:

Another aspect of the invention is the regulation of porcine leukocyte-type 12-LO mRNA in PVSMC cultured in normal (5.5 mM, NG) or high 25 mM, HG) glucose using quantitative RT-PCR. Figure 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 ³²P-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) . Densitometric analysis revealed nearly a 20-fold greater 12-LO expression in high glucose. In addition, experiments were performed to study the regulation of 12-LO mRNA expression by All (10~⁷M) at 24 hours using RT-PCR. Figure 7 shows that 12-LO mRNA expression 333 bp is much greater in high glucose (with little basal expression in normal glucose) . In addition, All caused a significant 3-4 fold increase in expression in both normal and high glucose. These results represent the first demonstration of regulation of 12-LO mRNA and indicate that glucose and All regulate 12-LO protein expression at the transcriptional level. The size of the transcript (4.0 kb) was confirmed using Northern analysis (data not provided) . II. Effects on Hypertrophy and Hyperplasia

Effects on hypertrophy: 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 10~⁸M. 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. In addition, Figure 10 shows that the 12-LO product 12-HETE could directly increase protein synthesis with the same potency as All in normal glucose. Moreover, 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) .

Effects on hyperplasia: Growth curves in PVSMC in 5.5 mM and 25 mM glucose are shown in Figure 12. The proliferation rates were approximately 30% faster in cells grown in elevated glucose. Moreover, in Figure 20 it is seen that the specific 12-LO inhibitor baicalein (10~⁶M) attenuated the growth responses suggesting that LO product formation may play a role in the proliferative response.

The effect of All alone and with LO inhibition on DNA synthesis as determined by [³H] thymidine incorporation has been measured. All caused a small but significant increase in DNA synthesis in normal glucose. The 12-LO inhibitor, baicalein, blocked All-induced DNA synthesis (Figure 9) , suggesting that products of the 12-LO pathway may mediate in part, All-induced proliferative effects. All-induced DNA synthesis was also enhanced in elevated (25 mM) glucose (normal glucose, 14617% vs high glucose 17418% control, p<0.05) (data not shown).

Therefore elevated glucose enhances basal proliferation and also enhances All-induced proliferative responses. In addition, blockade of the 12-LO pathway can reduce the proliferative actions of glucose and All.

Ill. Mechanism of Action of LO Products

Table 1 shows the results of Protein Kinase C (PKC) activity measurements in PVSMC grown in normal and high glucose. TABLE 1

The Effect of Various Agents on PKC Activity in PVSMC (Normal or High Glucose)

It is seen that in comparison to normal glucose, cytosolic activity in high glucose is lower, while membrane activity is higher indicating increased PKC activity in high glucose. Cells were treated with agents for 15 minutes. All alone showed only slight activation of PKC relative to TPA. However, in combination with 13- or 9-HODE, All showed increased activity in membrane fraction with reciprocal decrease in cytosolic activity. Immunoblotting experiments show that PVSMC express α and e isoforms of PKC but not β or forms.

These results show that cells cultured in high glucose show increased PKC activity and All can induce higher PKC activity in combination with LO products. Therefore, increased activity of certain

PKC isoforms may be a key mechanism for vascular cell proliferation in response to glucose All and the LO products.

IV. Studies in Human Adrenal, Mononuclear

Vascular Smooth Muscle and Endothelial Cells

Previously published studies show that All induced aldosterone synthesis in rat and human adrenal glomerulosa cells is mediated by activation of a 12-LO pathway (2,3) . This application presents new evidence that the particular isoform of 12-LO in human glomerulosa cells is a "porcine leukocyte type". Figure 13 shows the effect of All (10~⁷M) 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) . Thus, the 12-LO protein is present in cultured human glomerulosa cells as seen using an antibody against a porcine leukocyte 12-LO. Furthermore, the 12-LO protein expression is increased in cells cultured in the presence of All for 30 hours. A: shows immunoblot of data while B: represents a densitometric analysis of the data in A.

In Figure 14, the identification of the RNA for this form of 12-LO in human glomerulosa cells and mononuclear leukocyte type cells (U937 cells) can be seen using the previously described PCR assay, which is specific for this form of 12-LO. 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.

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 10~⁷M 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 10~⁷M All. Samples in lanes 4 and 5 were treated with RNase A prior to the RT-PCR. Lane 3 is without RNA. These results show that 12-LO gene is present in human glomerulosa and monocytes and that the RNA is upregulated by All.

To confirm that the amplified PCR product in human monocytes and adrenal cells is 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.

TABLE 2

Comparison of nucleotide sequences of porcine leukocyte 12-LO

(pl2-L0) cDNA and human adrenal 12-LO (HA12-L0) cDNA at the region between position 199 and 437 of published porcine leukocyte 12-LO cDNA seqeunce (14) . 2 nucleotide differences are at position 255 (T/C) and position 267 (C/T) .

HA12 0 AAACGGCACCTCCTTCAGGATGACGCGTGGTTCTGCAATTGGATCTCCGTGCAGGGTCCGGGAGCAAACGGG

I M 11 II I M i 111111 M M 11 M 111 M ! I ! M I ! 111 ! ! M 11 ! 11 ! ! 111 M III

P12L0 AAACGGCACCTCCTTCAGGATGACGCGTGGTTCTGCAATTGGATCTCCGTGCAGGGCCCGGGAGCAAATGGG 200 210 220 230 240 250 260 270

HA12LO GACGAGTTCAGGTTCCCCTGCTACCGCTGGGTGGAGGGCGACCGCATCCTGAGCCTCCCTGAGGGCACTGCC

P12LO GlAlClGlAlGlTlTlClAlGlGlTlTlClClClClTlGlClTlAlClClGlClTlGlGlGlTlGlGlAlGlGlGlClGlAlClClGlClAlTlClClTlGlAlGlClClTlClClClTlGlAlGlGlGlClAlClTlGlClCl

280 290 300 310 320 330 340

I

HA12L0 CGCACAGTGGTCGATGACCCTCAAGGCCTGTTCAAGAAACACAGGGAGGAGGAGCTGGCAGAGAGAAGGAAG

P12LO CGCACAGTGGTCGATGACCCTCAAGGCCTGTTCAAGAAACACAGGGAGGAGGAGCTGGCAGAGAGAAGGAAG 350 360 370 380 390 400 410

HA12L0 CTGTATCGGTGGGGTAACTGGAA (SEQ ID NO. 9)

P12LO CTGTATCGGTGGGGTAACTGGAA (SEQ ID NO. 10) 420 430

Furthermore, 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 (B₂ 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.

These studies confirm that a new gene has been found in several human tissues which encodes a 12-LO which is distinct from the one already, found in human platelets. These studies indicate that this 12-LO gene and protein is present in human adrenal, mononuclear and aortic smooth muscle cells and that All markedly upregulates both protein and RNA expression of 12-LO in several of these tissues (Figures 13, 16, 17 and 18).

In further studies using RNA from human aortic endothelial cells and the RT-PCR procedure described a leukocyte 12-LO was found to be expressed in these cells. 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, 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. Using 15-LO specific primers and probes (SEQ ID. 1-3 respectively) revealed specific amplification of the 15-LO cDNA used as a template. However, in two separate experiments no 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.

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.

No agent has yet been developed with the indication of blocking the activity or formation of this 12-LO pathway for prevention of hypertensive and diabetic vascular and renal disease. Several compounds are available for in vitro use that do block 12-LO activity. However, none have been developed for clinical use. Increasing evidence suggests that All and elevated glucose are each factors involved in accelerated vascular and renal disease. In addition, the mechanisms for increased atherosclerotic cardiovascular and renal disease in patients with diabetes remains unknown. The invention described here is apparently the first therapy designed to reduce or prevent a critical pathway involved in these disorders.

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(A) NAME: Irons, Edward S.

(B) REGISTRATION NUMBER: 16,541

(C) REFERENCE/DOCKET NUMBER: None (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 783-6040

(B) TELEFAX: (202) 783-6031

(C) TELEX: None

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown

(vii) IMMEDIATE SOURCE: Synthetically produced (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

AACTCAAGGT GGAAGTACCG GAG 23

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown (vii) IMMEDIATE SOURCE: Synthetically produced (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ATATAGTTTG GCCCCAGCCA TATTC 25

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown

(vii) IMMEDIATE SOURCE: Synthetically produced (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

AGGCTCAGGA CGCCGTTGCC C 21

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown

(vii) IMMEDIATE SOURCE: Synthetically produced (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

TTCAGTGTAG ACGTGTCGGA G 21

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 (B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown

(vii) IMMEDIATE SOURCE: Synthetically produced ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

ATGTATGCCG GTGCTGGCTA TATTTAG 27

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown

(vii) IMMEDIATE SOURCE: Synthetically produced (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCAGGATGCG GTCGCCCTCC AC 22

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 239

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown

(vii) IMMEDIATE SOURCE: Synthetically produced

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

AAACGGCACC TCCTTCAGGA TGACGCGTGG TTCTGCAATT GGATCTCCGT 50 GCAGGGTCCG GGAGCAAACG GGGACGAGTT CAGGTTCCCC TGCTACCGCT 100 GGGTGGAGGG CGACCGCATC CTGAGCCTCC CTGAGGGCAC TGCCCGCACA 150 GTGGTCGATG ACCCTCAAGG CCTGTTCAAG AAACACAGGG AGGAGGAGCT 200 GGCAGAGAGA AGGAAGCTGT ATCGGTGGGG TAACTGGAA 239

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 239

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Unknown

(vii) IMMEDIATE SOURCE: Synthetically produced

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

AAACGGCACC TCCTTCAGGA TGACGCGTGG TTCTGCAATT GGATCTCCGT 50

GCAGGGCCCG GGAGCAAATG GGGACGAGTT CAGGTTCCCC TGCTACCGCT 100

GGGTGGAGGG CGACCGCATC CTGAGCCTCC CTGAGGGCAC TGCCCGCACA 150

GTGGTCGATG ACCCTCAAGG CCTGTTCAAG AAACACAGGG AGGAGGAGCT 200

GGCAGAGAGA AGGAAGCTGT ATCGGTGGGG TAACTGGAA 239

Claims

CLAIMS :

1. Isolated and purified 12-LO expressed by human vascular smooth muscle cells or human adrenal cells or mononuclear cells or endothelial cells, said isolated and purified 12-LO being distinct from that found in human platelets and distinct from human 15-LO.

2. A method for treating human vascular disease which comprises inhibiting the expression of the human 12-LO as defined by claim 1 to mediate angiotensin II and glucose induced vascular and renal actions.

3. A method as defined by claim 2 in which said mediation is accomplished by administration of a 12-LO inhibitor—e.g. , baicalein.

4. Isolated and purified human 12-LO RNA from human vascular smooth muscle cells or adrenal cells or mononuclear cells or endothelial cells, said isolated and purified 12-LO being distinct from that found in human platelets.

5. Human leukocyte 12-LO.

6. An isolated purified or substantially purified nucleotide sequence which consists essentially of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, or 8.

7. A method which comprises regulating the protein and RNA expression of the human 12-LO defined by claim 1 by culturing said cells iji vitro in a medium in which the concentration of angiotensin II is controlled.

8. A method as defined by claim 7 in which the concentration of angiotensin II is increased to upregulate expression of 12-LO protein and RNA or decreased to down regulate expression of 12-LO protein and RNA.

9. A method for mediating angiotensin II and glucose induced vascular and renal action by controlled activation of the expression of 12-LO expressed by human vascular smooth muscle or adrenal or endothelial or mononuclear cells.

10. A method as defined by claim 9 in which said activation is controlled by regulating the level of ambient glucose concentration bathing said cells.