Dear colleagues,

In the beginning of my presentation, I would like to thank the Department of Pharmacology for the opportunity to present my results. We know angiogenesis not only as growth of new blood vessels. It is also an important component of many pathological conditions such as tumor growth. Similarly, abnormal growth of new blood vessels has been observed for many serious eye diseases like diabetic retinopathy, age-related macular degeneration and certain inherited retinal degenerations.

In Dr. Schwartzman’s lab, we study biologically active arachidonic acid metabolites. I study 12(R)-HETrE, which is one of the most potent proinflammatory and proangiogenic compounds synthesized by the eye. We work in three different areas (slide 1). First, we explore signaling mechanisms, by which 12(R)-HETrE may affect the cell. Secondary, we work the gene regulation of cytochrome P450 4B1 isoform, which is one of the enzymes involved in the development of the inflammatory response. Third, we try to identify and clone the specific receptor of 12(R)-HETrE.

RESEARCH AREA 1: I would like to remind that inflammatory response in the eye follows an epithelial injury. There are three major symptoms of the eye inflammation: conjunctival vasodilation, edema and neovascularization. Two first symptoms are reversible. Neovascularization may or may not be reversible depending on the degree of vascular invasion. Vascularization becomes a serious clinical problem since physical presence of new vessels makes a complication to the sight. If vessels do not completely regress, it may result in permanent loss of visual acuity.

N.B!: [Conjunctival vasodilation[AM1]] [Edema[AM2]] [Neovascularization[AM3]]

Inflammation of cornea accompanies by a massive release of eicosanoids. Eicosanoid profile, ratio and content are tissue- and organ specific. They also depend on physiological conditions. Cornea responds to the injury by the synthesis of two eicosanoids: 12-HETE and 12(R)-HETrE. The first compound, 12-HETE, increases corneal thickness and reduces intra-ocular pressure (IOP). It is also known as an inhibitor of Na, K-ATPase and neutrophil chemoattractant. Another one, 12(R)-HETrE exhibits potent inflammatory and angiogenic properties.

N.B!: [12-HETE[MLS4]] [12(R)-HETrE[MLS5]]

Pro-inflammatory and angiogenic role of 12(R)-HETrE is strongly supported by our earlier study: 1) Synthesis of 12(R)-HETrE is increased and followed the injury, 2) It positively correlates with the severity of inflammatory response, 3) The biological activities of 12(R)-HETrE include vasodilation, neutrophil chemotaxis and angiogenesis, which are typical for proinflammatory factors, 4) 12(R)-HETrE is present in human tears as well as its level is much higher in tears from subjects with ocular inflammation.

We isolated rabbit limbal microvessel endothelial cells from corneal explants by the selection the cells on extracellular matrix in the presence of specific medium. Stimulation of cells with 12(R)-HETrE resulted in formation of pseudocapillary that we were able to scoop out from the gel and reseed on fibronectin in ordinary cell culture flasks.

By the fact that selection occurred due to the presence of 12(R)-HETrE, we can already prove that the cells are sensitive to 12(R)-HETrE. Then, we cultivated cells the flasks and split them after they covered about 70-80% of total growth surface and changed medium every second day. Thirty-six hours before the experiment, we changed medium for a basal one supplemented with 1% FBS and antibiotic and continued to cultivate them. This time, 36 h, is enough to stop a visible cell growth and get cells prepared for the treatment.

Because of synthesis of 12(R)-HETrE is induced in response to hypoxic injury, we examined its possible relation with vascular endothelial growth factor (VEGF). As you can see from this slide, treatment the cells with 12(R)-HETrE increased VEGF mRNA levels in a time-dependent manner. We observed 5-fold increase over the control in 45 min after the treatment. Further co-incubation resulted in gradual decline of mRNA level, which became comparable to control in 48 h.

Incubation of cells with cycloheximide did not affect VEGF mRNA level in treated cells (lane 4), suggesting that the induction does not require de novo protein synthesis. On the other hand, addition of actinomycin D abolished 12(R)-HETrE-induced VEGF expression, indicating that this effect requires de novo RNA synthesis.

[Actinomycin D[AM6]] [Cycloheximide[AM7]]

[Necessity of “de novo” protein synthesis for mRNA production[AM8]]

To determine if treatment with 12(R)-HETrE affected RNA stability, we measured VEGF mRNA levels in the presence of actinomycin D in cells treated with 12(R)-HETrE or vehicle. The half-life of VEGF mRNA was about 9 h in the absence of 12(R)-HETrE and 14 h in the presence of 12(R)-HETrE, suggesting that 12(R)-HETrE affected somehow both transcriptional activation and mRNA stabilization.

12(R)-HETrE also increased VEGF mRNA in concentration-dependent manner with maximal effect at 0.1 nM. This effect was also stereospecific because S- enantiomer did not significantly affect VEGF expression at concentrations of up to 10 nM. We measured VEGF mRNA levels at different concentrations of 12(S)-HETrE and found that it barely exceeded control level[AM9].

[Stereospecificity[AM10]]

Induction of VEGF expression by 12(R)-HETrE might occured through the activation of MAPK kinases. In the cell, mitogen- activated protein kinases (MAPKs) contribute in a variety of cellular responses to growth factors, hormones and cytokines. This group of enzymes is also involved in cell proliferation, cell migration, apoptosis and neovascularization. Moreover, some arachidonic acid metabolites enhanced p42/p44 phosphorylation. We examined if 12(R)-HETrE-induced VEGF expression due to activation of the ERK. After the addition of 12(R)-HETrE, both isoforms ERK1 and ERK2 were transiently activated. You can see Western blot analysis with antibody against the phosphorylated forms of ERK1/2. Kinase activation peaked at 5 min and gradually decreased to control levels by 60 min.

In vitro kinase assays also demonstrated an increase in phosphorylation of Elk, the specific substrate of ERK, which paralleled the increase in ERK phosphorylation. The profile repeated one for ERK phosphorylation.

This slide shows that preincubation of cells in the presence of ERK specific inhibitor, PD98,059 significantly affected effect of 12(R)-HETrE. In this slide, lane 1 was a positive control (the recombinant ERK provided with the Kit). Lane 2 was cell treated with vehicle; lane 3- cells activated with 12(R)-HETrE and #4 illustrated that PD compound prevented activation by 12(R)-HETrE.

Northern blot analysis indicated the presence of two VEGF transcripts with estimated sizes of 2.4 and 4.2 kb. 12(R)-HETrE induced expression of both transcripts in a concentration-dependent manner[AM11].

This slide demonstrated that besides an effect on ERK the PD compound also affected VEGF mRNA production suggesting a cause relationship between 12(R)-HETrE treatment, ERK activation and VEGF synthesis. Same as two slides back “PD” prevented effect of 12(R)-HETrE. This time it affected VEGF mRNA synthesis.

VEGF mRNA synthesis in response to 12(R)-HETrE was followed by protein levels: In the medium of the cells treated with 0.1 nM 12(R)-HETrE VEGF secretion increased in a time-dependent manner. We observed a significant increase in 3 h after the tretment and a 4-fold increase at 6 h after the addition of 12(R)-HETrE.

The effect of 12(R)-HETrE on VEGF secretion was also concentration-dependent. While it was difficult to say of the position of maximum. It was likely expect it also at 0.1-1.0 nM range.

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Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea after hypoxic injury, their interaction may be an important determinant in the development of neovascularized tissues.

At the final step of our study we performed the in vitro capillary tube-like tube formation assay. We wanted to estimate angiogenic potential of 12(R)-HETrE on the endothelial cells. As seen in the following slide (Fig. 10), 12(R)-HETrE stimulated cell morphogenesis at a concentration as low as 1 pM. This effect was concentration-dependent, with the maximal stimulation achieved at 1 nM. This effect was also stereospecific. Unlike 12(R)-HETrE, S enantiomer had little effect on tube-like capillary formation at concentrations up to 10 nM (Fig. 11). Importantly, the addition of anti-VEGF antibodies to the incubation medium greatly attenuated pseudocapillary formation (Fig. 12). That indicated that 12(R)-HETrE effects on the cells are mediated, at least in part, by VEGF.

Fig. 10

Fig. 11

Fig. 12

Instead of conclusion of this part I would like to show the following diagram that briefly illustrates the events happening in the cells after treatment with 12(R)-HETrE:

A) 5 min after treatment, the signal goes down through MAPK cascade. Phosphorylation of MEK1/2 reaches the maximum B) 15-45 min after treatment, VEGF mRNA level rises, At 1h, the level of phosphorylated ERK declines, C) 3-4 h after the treatment, secretion of VEGF protein follows VEGF mRNA rise, D) 4h: cell morphogenesis starts, E) 13 h about a half of newly synthesized VEGF mRNA is now destroyed F) On the other hand, cell morphogenesis reaches the maximum. Cells actively interact each other and form pseudocapillary network on the Matrigel G) In 48 h, level of VEGF mRNA decreases to control value. Pseudocapillary network thinner and finally disappears H) After 48 h, cells apoptosis kills the cells. Some cells will be alive. However, their amount will decrease swiftly.

RESEARCH AREA 2 Another part of my research program is concentrated at gene regulation of CYP 4B1. We hypothesized that CYP 4B1 is involved in the production of proinflammatory and angiogenic eicosanoids, particularly 12(R)-HETrE. In general, eicosanoids can be produced by three different groups of enzymes: cyclooxygenases (COX), lipoxygenases (LOX) and cytochrome P450 monooxygenase (CYP). Our group established the importance of CYP-dependent pathway in initiation and development of inflammatory response in the eye. We also showed a link between severity of the inflammatory response and synthesis of 12(R)-HETrE. We demonstrated that inhibition of CYP sufficiently decreased 12(R)-HETrE content in the tissue. It also attenuated ocular surface inflammation suggesting a potential cause-effect between these events.

We also isolated and cloned a corneal epithelial CYP4B1 full-length cDNA. Here is the strategy for the cloning of cDNA. We used TRIZOL method for isolation of RNA. This step was followed by RT- reaction, which followed by PCR amplification. One of the primers for PCR was universal amplification primer (oligi dT) and another one was a custom designed gene specific primer. Both primers carried adaptor sequences with restriction sites. Then we prepared DNA for cloning: on 5’-side we generated AccI site, on 3’- NotI site. Finally, we cloned cDNA in Bluescript vector linerized with NotI and AccI.

Earlier we hypothesized that CYP 4B1 might underly the synthesis of proinflammatory eicosanoids in response to hypoxic injury. For better understanding the molecular regulation of CYP 4B1. we isolated and cloned the CYP4B1 promoter. We constructed DNA libraries from rabbit corneal epithelial genomic DNA, linked the adaptor. The primary PCR we performed with gene specific primer and adaptor-specific primer. A site for ASP was not originally present. It appeared in the DNA only after amplification with GSP (28 n). Combination this strategy with touch down PCR method supposed to increase specificity of amplified products. Then we performed nesting PCR with two other sets of primers and isolated two overlapping fragments with Mw 0.65 and 3.4kb. Then, we cloned products in bluescript and sent the results for sequencing.

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We analyzed the results by computer software (DNA star, Lasergene) for the presence of known cis-acting elements. The analysis showed the presence in the promoter of DNA binding sequences for a viriety of known transcription factors. Some of them such as HIF-1, NFkB and AP-1 may activate gene expression in response to hypoxia.

HIF

Then we checked the results by Electrophoretic mobility shift assay (EMSA). In this slide you can see the induction of HIF-1 binding with a probe from the cloned CYP4B1 in the cells exposed to hypoxia. We confirmed the specificity with a competition assay as well as supershift assay with anti-HIF sntibody.

The nuclear binding activity of AP-1 and NFkB probes from the CYP4B1 promoter was also enhanced in response to hypoxia suggesting that these transcription factors contribute to the hypoxic induction of CYP4B1 expression.

Conclusion for Part 2:

The results of this study provide the first molecular mechanistic explanation for the induction of CYP4B1 and, thereby, the production of inflammatory eicosanoids in response to hypoxic injury. We would continue this study and fully evaluate the molecular regulation of this gene during inflammation.

Perspectives:

Currently we continue to study CYP 4B1 regulation and perform transfection experiments on rabbits in vivo. For these experiments we developed a tattoo technique, which consists of multiple microinjections with a plasmid made around the eye limbus. The transfection is followed by 3 days incubation period. Then we sacrify animals and analyze of gene expression with different techniques such as Matrigel assay, HPLC or immunohistochemistry. Because of lock of the time am not ready to present these data today. Hopefully they will be ready for a next time.

RESEARCH AREA 3

Typically a cell- signaling mechanism starts from the binding of a ligand to its specific receptor. This initial step is very important for the whole metabolic pathway. If appropriate information from the environment won’t be transduced, the final result will be diminished and cell the vessels won’t grow. However, if conditions allow transducing the signal, it will multiply and moving downstream this signal will cause the effect. The specific primary mediator- receptor interaction may be an attractive therapeutic approach for the treatment of many ocular diseases including diabetic retinopathy, age-related macular degeneration and certain inherited retinal degenerations. Identification and cloning the receptor will obviously open a door to creation synthetic receptor antagonists capable selectively inhibit angiogenesis and inflammatory response via abolishing the signal transduction.

	We developed the following strategy for identifying and cloning of the receptor. In the beginning we would analyze nearest structural analogs of 12(R)-HETrE for their ability to induce angiogenesis in vivo and RLME morphogenesis- in vitro. We would identify the potential agonists and antagonists of 12(R)-HETrE. Then, we would confirm the mechanism for the analogs with the strongest effect. At this step, the data can be used for computer modelling of the specific receptor 12(R)-HETrE binding site. Moving forward, next we would isolate the receptor due to purification of a dead-end complex of the receptor itself and the strongest of analogs. Our goal at that step is to get N-terminal protein sequence and make the specific antibody. With antibody we will be able to scale up the method for protein purification and obtain the receptor in quantities enought for the sequencing. This second sequencing (50-60 terminal aminoacids) will give us a chance to find a sequence of interest in a Gene Bank and proceed to the routine cloning of the receptor cDNA.
In cooperation with Dr. J.R. Falk (the University of Texas Southwestern Medical Center at Dallas, Texas) we designed and analyzed more than 20 different synthetic analogs of 12(R)-HETrE. We demonstrated the ability of few of them to interfere with 12(R)-HETrE angiogenic effect. Few months ago, we started a new study that should estimate anti-angiogenic and anti-inflammatory potential of these perspective compounds on rabbits.		(list of tested compounds with names and structures)

Due to their strong and specific binding to the receptor, the receptor antagonists are very powerful tool for purification of the receptor. Our previous works already gave us very promising results. We demonstrated a presence of a 12(R)-HETrE binding site in the corneal endothelial cell. We showed that this binding site has high affinity to 12(R)-HETrE (~0.1 nM). We propose that this binding site serves as 12(R)-HETrE specific receptor and is responsible for the 12(R)-HETrE signal transduction in the corneal endothelium.

Stoltz R., Schwartzman ML 1997, JOPT, 13, P. 195

Use of receptor antagonists with therapeutic goal is one of the most hopeful approaches of modern medicine. We expect that similarly to other receptor antagonists 12(R)-HETrE receptor antagonists will suppress angiogenesis and inflammatory response due to prevention of 12(R)-HETrE binding to the receptor. Another practical approach for the receptor studies would be development of new treatments for the genetic therapy of vascular diseases. Knowledge of the receptor sequence and structure would help to design its modified form that would carry 12(R)-HETrE- binding domain but would be deficient in the signal transduction function. The introduction of this modified receptor to the cell will eliminate of 12(R)-HETrE from the circulation flow delaying the development of vascularization. The results of this work will also aid in the development of safe and selective receptor antagonists useful for the treatment of eye disorders and help to improve our knowledge of the 12(R)-HETrE role in human physiology.

CONCLUSION

Ocular neovascular diseases afflict the vast majority of patients who suffer from catastrophic loss of vision. Age-related macular degeneration (ARMD) affects 12-15 million Americans over the age of 65 and causes visual loss in 10-15% of them as a direct effect of choroidal neovascularization in the eye. The leading cause of visual loss for Americans under the age of 65 is diabetes; 16 million individuals in the United States are diabetic and 40,000 per year suffer from ocular complications of the disease, often a result of retinal neovascularization. While we are entering a new era in terms of medical treatment of eye diseases with a neovascular component, it is important to remember that abnormal, new blood vessel growth is the only one pathological manifestation of the underlying diseases.

* In the case of macular degeneration, we still need to address the primary disorder that leads to choroidal neovascularization

* In the case of ischemic retinopathies such as diabetes, we still not have cured the underlying disease that leads to vascular insufficiency and associated hypoxia.

These remain distant, but hopefully attainable, goals. The real challenge in treating vascular diseases is to better understand the molecular and cellular mechanisms regulating angiogenic processes. Another area of challenge is to design effective drug(s), which selectively, potently and in physiologically meaningful doses, deliver drugs to the affected area of the eye. The finding of significant amounts of 12-HETrE, a potent angiogenic eicosanoid, in tears from inflamed human eyes underscores the importance of understanding this eicosanoid synthesis, regulation and mechanism of action. As a scientist, I am very impressed by the power of angiogenic process. I am very proud with the feeling that I can make my own contribution in the fight with sight loss and blindness.

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In the end of my presentation I would like to thank my mentor Prof. Schwartzman, my colleagues in the lab, all people who made a valuable contribution in this project. Dr. Mastyugin (Volodia) and Mr. Wen Xiang made the most of routine work related to cloning. Wen Xiang with Silvia Ashkar, MD also performed some experiments with the promoter constructs. Silvia also established most of techniques and protocols for the eye surgery. I also cannot forget of Mrs Seta. Franceska and I continue to study 12(R)-HETrE signaling mechanism in endothelial cells.

an abnormal infiltration and excess accumulation of serous fluid in connective tissue or in a serous cavity

growth of new blood vessels

12-hydroxy-5,8,10,14-eicosatetraenoic acid

12-hydroxy-5,8,14-eicosatrienoic acid

Metabolic inhibitor; inhibits mRNA synthesis (polII)

Inhibitor of eucariotic peptidyl transferase. Binding of CH to the enzyme prevents translocation

Synthesis of VEGF mRNA occurs without “a middle man”. “Middle man” is a gene, which expression might be required for the expression of ours. In other words, cell already has all required proteins to express VEGF

Data for 12(S)-HETrE: 115 ± 26%, 65 ± 31%, and 99 ± 22% of control levels at 0.1, 1, and 10 nM 12(S)-HETrE

If one of side groups in the molecule may have opposite symmetric orientation to the flat made by its neighbor side groups we talk of stereoisomers. We used to call them “R” and “S”. Due to different orientation of the 12th hydroxyl group 12(R)- and 12(S)-HETrE have different properties

Sorry for not presenting housekeeping blot for this membrane. I cannot find it.