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Specific Aims
The pathologic hallmark feature of accelerated vascular disease, such as restenosis following intravascular interventions, is the formation of a neointima composed of hyperplastic smooth muscle cells (SMCs). Heparin-like compounds are necessary to achieve homeostasis in vascular tissue, and injury to the endothelium lining of a blood vessel disrupts the balance of these and other compounds that regulate surrounding tissue. Heparin and heparin-like molecules have been shown to inhibit SMC proliferation in vitro and in vivo. Protamine, a chelator of heparin, is used clinically to reverse heparin's anticoagulative effect and also in some common insulin preparations. Protamine has been shown to reverse heparinıs antiproliferative effect on vascular SMCs. Because diabetic patients are at high risk of developing vascular disease, there is a potential danger of using such compounds. Thus far, the mechanism by which protamine negates the inhibitory effect of heparin has not been determined. Our proposal is aimed toward elucidating this mechanism.
Our central hypothesis is that protamine-heparin complexes change size and charge in a time-dependent manner, as the complexes reconfigure to lower energy states. Thus, more than merely chelation of the compounds occurs and accounts for the reversal of heparin's antiproliferative effect on the SMCs.
In order to simplify this problem, the effects of protamine on heparin will be examined initially. Later, the studies will be expanded upon to test the effects of protamine on heparin-like species found in vascular tissues, namely heparan sulfate.
We have the following specific aims proposed seeking to verify our central hypothesis:
1. Observe the change in the protamine-heparin complexes over time. Protamine and heparin complexes are visible under magnification. Therefore, protamine and heparin will be incubated with and without SMCs and the morphology of the complexes will be observed. Changes in the appearances of the complexes over time will suggest that the complexes change conformations.
2. Look for evidence suggesting that substances released by the smooth muscle cells bind the protamine/heparin complexes. We will determine if there are differences in the appearance of the complexes when in the presence of SMCs and in cell-free systems. Differences will suggest that substances from the cells are interacting with the complexes.
3. Ascertain whether or not protamine/heparin complexes have a time-dependent effect on SMC growth. The effects of preincubating protamine and heparin solutions for 24 hours before adding them to SMCs versus adding the solutions without incubation will be tested. If the amount of proliferation is different, this will support the idea that over time the complexes have different sizes and charges, and thus have different effects on the cells.
4. Determine whether or not protamine prohibits heparin from being internalized or alters the amount of heparin internalized. Using labeled heparin, we will quantify the amount of heparin outside, on the surface, and inside of the SMCs with and without protamine. If the amount of heparin internalized is less in the presence of protamine, this may suggest that protamine alters heparin's transcriptional regulatory functions by preventing internalization of the compound.
5. Compare how protamine affects heparan sulfate and heparin. We plan to compare smooth muscle cell growth in the presence of protamine and either heparin or heparan sulfate. We also plan to determine if heparan sulfate-protamine complexes are formed in the same way that protamine and heparin form complexes, and if the complexes have the same appearance both with and without smooth muscle cells.
As a result of the above studies, we aim to better understand the way in which protamine and heparin interact and affect the growth of SMCs.
Background and Significance
The blood vessel wall is composed of three layers: the intima, which lines the vessels and consists of an endothelial cell monolayer called the endothelium; the media, which is made up of multiple layers of SMCs; and the adventitia, which is made up of fibroblasts and loose connective tissues. An intact endothelial layer is necessary to achieve vascular homeostasis. The endothelium secretes a number of substances, the sum of which helps regulate smooth muscle cells. in vitro , endothelial cell conditioned medium (ECCM) from confluent endothelial cells (ECs) inhibits SMC growth, while ECCM from sparse cultures of ECs stimulates SMCs. In vivo, disruption of the arterial endothelial layer, by interventions such as balloon angioplasties, causes smooth muscle cells to proliferate and results in the formation of a neointima.
Heparin-like substances that inhibit smooth muscle cell growth are produced by endothelial cells (1) and smooth muscle cells (9). Both in vitro and in vivo, exogenous heparin inhibits SMC growth (3,5); heparan sulfate secreted by endothelial cells may account for much of the inhibition of SMCs (10).
Clinically, protamine is administered to reverse heparin's anticoagulative properties. Protamine is also used as part of common insulin preparations to prolong the uptake of insulin by decreasing its solubility in the blood. Protamines are a family of basic proteins rich in arginine. They are purified from fish sperms and reverse the anticoagulative effect of heparin by establishing 1:1 pairing of its cationic sites with the anionic heparin sites (7). Protamine has been shown to inhibit the ability of mitogenic factors such as acidic fibroblast growth factor (aFGF) (17), basic fibroblast growth factor (bFGF) (17), and platelet derived growth factor (PDGF) (11) to stimulate proliferation in vitro by preventing these growth factors from binding to their cell surface receptors. In contrast, protamine potentiates the mitogenic action of epidermal growth factor (EGF), possibly by altering the binding of reactants (12). Although protamine has been shown to reduce the activity of many growth factors, and potentiate the activity of only EGF, it has been found to have a stimulatory effect on vascular SMCs in culture and to interfere with the growth inhibition heparin causes in SMCs in vitro and in vivo (8). Furthermore, the inhibitory effect of conditioned medium from post-confluent endothelial cells is reversed by protamine (10).
To our knowledge, the action by which protamine reverses the antiproliferative properties of heparin has not been identified. Heparin regulates vascular repair in a variety of ways, and protamine may interrupt this regulation by interfering with any number of these effects. Heparan sulfate proteoglycan (HSPG) binds and sequesters growth factors, and heparin-like compounds block the binding of heparin-avid growth factors PDGF, aFGF, and bFGF. By chelating HSPG or these heparin-like compounds, protamine may reverse heparin's antiproliferative effect on SMCs by increasing the growth factor activity. Heparin also affects gene expression, including suppressing c-fos and c-myc gene expression by inhibiting a protein kinase C-mediated pathway (18, 20). By complexing with protamine, heparin may be prevented from being internalized and therefore may not perform this function. Alternatively, protamine may work by some direct mechanism of its own; it has been shown to potentiate a growth factor without heparin avidity, EGF.
In work by Han et al. studying vascular SMC inhibition by ECCM (10), the addition of protamine reversed the growth inhibition caused by ECCM, providing evidence that the inhibition occurred at least in part due to the presence of HSPG or other heparin-like compounds in the conditioned medium. However, in this work, high doses of protamine (above 20 µg/ml) administered with ECCM stimulated cell growth above and beyond the administration of protamine alone. This result is thus far unexplained and provides evidence that more than merely chelation of HSPG is occurring.
Studies of heparin and protamine interactions indicate that initially 1:1 binding of cationic and anionic sites occurs, but that over time there is further binding of heparin, presumably due to the different affinities of the sulfate and carboxyl groups of heparin (6). These complexes were found to change size and shape over time, as heparin's sulfate groups preferentially bind to the complex and release some of the bound carboxyl groups. This may indicate that over time heparin's antiproliferative activity decreases in the presence of protamine. Studies have shown that heparin's sulfate groups are important for its inhibitory effect on SMCs (2, 19). With this in mind, we believe that protamine-heparin complexes will have different effects on SMC growth as the compounds have been incubated together for longer periods of time, as the shape and charge of the complexes are time-dependent.
These observations bring into question the clinical use of protamine to reverse systemic heparin administration and as part of routinely used insulin preparations. In addition to its possible role in exacerbating vascular disease, there has already been concern that protamine causes hypotension and vascular collapse (13, 15, 16). Its use after procedures such as bypass surgery or balloon angioplasty may be especially harmful. Exacerbation of vascular disease is also a large danger for diabetics who rely on protamine-insulins, as they are already at an increased risk for vascular injury.
Preliminary Results
To simplify our studies of the mechanism by which protamine reverses the inhibitory effect of heparin-like molecules, we began working with heparin and protamine. Our work so far has involved studying the complexes formed both in the presence of SMCs and in cell-free systems, and looking at the effects of protamine and heparin on SMC growth. Eventually, we hope to expand these experiments to heparan sulfate in order to more closely realize how protamine interacts with this substance secreted by ECs.
Specific Aim: 1. Observe the change in the complexes over time.
Methods:
Bovine arterial SMCs (5E3 cells per well, passage 6) were seeded in 24-well plates and incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% calf serum for 24 hours. The cells were then growth arrested for 72 hours by incubating the cells in 0.1% calf serum/DMEM. To release from growth arrest, the medium was replaced by 5% calf serum/DMEM, 5% calf serum/DMEM supplemented with various amounts of protamine and/or heparin, and 5% calf serum/DMEM with protamine and/or heparin that had been incubated together with the medium for 24 hours at 37°C. At this time, 5% calf serum/DMEM was also added to 24-well plates without SMCs, and protamine and heparin were added in the same amounts used with the SMCs. All of the 5% calf serum/DMEM added had been incubated at 37°C for 24 hours in the same manner as the medium with the protamine and heparin added. The protamine and heparin solutions were made with Grade X protamine sulfate (Sigma) or heparin sodium (Pharmacia Hepar) with a potency of 160 Units/mg (USP) and Ca++- and Mg++-free phosphate buffered saline (PBS) solution at concentrations of 1 mg/ml.
Results:
Initially, no complexes were visible after adding the medium with heparin and/or protamine solutions to the cell and cell-free systems. However, after three days of incubation, all wells containing both heparin and protamine had conglomerations of matter visible, with some dosages of protamine and heparin having larger formations than others. These conglomerations were caused by the presence of both heparin and protamine, as the wells without these compounds or with only one of these compounds did not contain such complexes. Also, mixing portions of the 1 mg/ml protamine and heparin solutions revealed similar structures under magnification. Thus, it appears that protamine and heparin complexes change size over time in both cell and cell-free systems.
Specific Aim: 2. Find evidence supporting the fact that substances released by the smooth muscle cells bind the protamine-heparin complexes.
Methods:
Photographs were taken of the complexes in each well from the above experiment three days after the addition of the solutions containing protamine and/or heparin.
Results:
The complexes visible in the presence of SMCs were found to be larger than the structures in cell-free medium for all amounts of protamine and heparin. Figures 1 through 6 illustrate these differences in sizes for two dosages of protamine and heparin: 40 µg/ml heparin sodium with 5 µg/ml protamine sulfate (Figures 1-3) and 40 µg/ml heparin sodium with 40 µg/ml protamine sulfate (Figures 4-6). These observations support the hypothesis that the protamine-heparin complexes bind to substances secreted by the SMCs. In addition, there were larger complexes in the presence of cells with preincubated protamine and heparin than in the presence of cells with solutions that were not preincubated (compare Figures 2 and 3, 5 and 6). Many of the wells with preincubated solutions had one or more large clumps of material in the center of the well, with smaller complexes dispersed around it (see Figure 6).
In addition to the observations from the previous section, these observations further support the idea that the protamine-heparin complexes change over time. The preincubated and non-preincubated solutions formed different conglomerations when incubated with SMCs. Because the complexes should be in different conformations if they have been preincubated for 24 hours, one would expect that they would bind different substances or amounts of substances from the SMCs. The observation that the complexes are different in cell and cell-free systems suggests that the complexes do bind substances from the SMCs.
Specific Aim: 3. Determine whether or not protamine-heparin complexes have a time-dependent effect on SMC growth.
Methods:
In order to test if the protamine-heparin complexes have different effects on SMCs at different times of preincubation, the cells in the above experiment were washed with Ca++- and Mg++-free PBS after three days of incubation in the 5% calf serum/DMEM solutions with and without protamine and/or heparin. The cells were then removed from the wells by trypsinization and cell number was determined using a Coulter counter.
Results:
Figures 7 and 8 show the results of the growth assay. Each data point is represented by the mean ħ the standard error for the experiment plated in triplicate at each dose. Percent inhibition is measured as:
100*[(cell count in 5% CS plus heparin and/or protamine ÷cell number in 5% calf serum) - 1]. A positive number therefore indicates stimulation, while a negative number indicates inhibition.
Our laboratory has been having trouble with the consistency and repeatability of experiments using bovine SMCs. However, comparing the experimental results from cells in non-preincubated protamine and heparin solution (Figure 7) with results from cells in preincubated solutions (Figure 8), there are different growth patterns even when the dosages were identical. On Figure 8, the non-preincubated, protamine-only series is included as a reference. The different trends in our preliminary results suggest that the complexes have effects on SMC growth in a time-dependent manner. These experiments will be repeated once the problems with SMCs are resolved in our laboratory.
Planned Experiments
Preliminary results indicate that the protamine-heparin complexes alter their conformation over time, and that the complexes affect SMC growth in a time-dependent manner. We plan to conduct studies similar to our preliminary work to verify our results. We also plan to conduct further experiments to determine if protamine may be reversing heparin's antiproliferative effect on SMCs by preventing heparin from being internalized or reducing the amount of heparin internalized. In addition, we hope to determine if protamine and heparin-like molecules secreted by ECs behave in the same manner as protamine and heparin. To our knowledge, there have not been studies comparing the interactions of protamine and heparin with the interactions of protamine and heparan sulfate. From these results, we can ascertain whether or not we should be studying heparan sulfate rather than heparin in order to more closely realize the effect protamine has on compounds secreted by blood vessels when it is used clinically.
Specific Aim: 1. Observe the change in the complexes over time.
Methods:
In order to test the size and conformation of the complexes over time, we will seed 24-well plates with bovine aortic SMCs in the manner described above, adding protamine and heparin solutions (both preincubated and non-preincubated) with 5% calf serum/DMEM to release them from growth arrest. We plan to monitor the size of the complexes over time carefully, using a data analysis program to quantify the amounts and sizes of the complexes at one, two, and three days after addition of the compounds.
Expected Results:
Based on our preliminary results, we expect this data to show that the complexes become larger over time, indicating that the protamine and heparin complexes alter their conformations.
Specific Aim: 2. Find evidence supporting the fact that substances released by the smooth muscle cells bind the heparin/protamine complexes.
Methods:
Using the above experiments and data analysis, we plan to compare the complexes in the presence of cells with the complexes in systems devoid of SMCs to determine if substances from the SMCs bind or alter the conformation of the complexes.
Expected Results:
Based upon our preliminary results, we expect the data analysis to indicate that the complexes visible in cell versus non-cell systems are different in their sizes and amounts. This would provide further evidence that substances from the SMCs bind the complexes.
Specific Aim: 3. Determine whether or not protamine/heparin complexes have a time-dependent effect on SMC growth.
Methods:
To determine if the protamine-heparin complexes affect the SMCs differently after being incubated together for different periods of time, the SMCs plated for the above experiments will be removed by trypsinization after 72 hours of incubation with the protamine and/or heparin compounds. Cell number will be determined with a Coulter counter.
Expected Results:
The problems we have been experiencing with the SMCs in our laboratory seem close to being resolved, so we believe that more reliable data can be now gathered. If the growth of the SMCs in the presence of the preincubated protamine and heparin solutions is different than the growth in the non-preincubated solutions, this will support our hypothesis that the complexes affect SMC growth in a time-dependent manner.
Specific Aim: 4. Determine whether or not protamine prohibits heparin from being internalized or alters the amount of heparin internalized
Methods:
We plan to seed SMC as described above (5E3 cells/well, growth arrest 24 hours), and to release from growth arrest by adding protamine and/or [H3]heparin with 5% calf serum/DMEM. After 72 hours of incubation, the media, cell-surface bound material obtained following incubation with a 100-fold excess of unlabeled heparin (19, 20), and internalized material will be collected and [H3]heparin quantified using liquid scintillation counting.
Expected Results:
If protamine prevents the amount of heparin from being internalized in the cells and providing transcriptional regulation, we expect that less heparin will be internalized as the amount of added protamine increases. These experiments will also allow us to determine if the cell-binding of heparin is changed in the presence of protamine.
Specific Aim: 5. Compare how protamine affects heparan sulfate and heparin.
Methods:
In order to compare protamine's effect on heparan sulfate with its effect on heparin, we plan to determine if visible complexes are formed when protamine is incubated with ECCM (which contains heparan sulfate), and if so, to monitor the sizes of the complexes over time in both in the presence of bovine aortic smooth muscle cells and in cell-free systems, similar to the manner described above. In addition, we plan to repeat the SMC growth assay using ECCM and various amounts of protamine sulfate, to see if the growth patterns are similar.
Collection of Endothelial Cell Conditioned Medium: Bovine aortic endothelial cells will be grown to confluency in 100 mm tissue culture dishes in 5% calf serum/DMEM, replacing the medium 72 hours after confluency is reached. After 24 hours, the conditioned medium will be collected and centrifuged at 1000 rpm, collecting the supernatant and storing aliquots at -20°C (10).
Testing for complexes from preincubation of protamine and ECCM with and without SMCs: Bovine aortic SMCs will be seeded similar to the above experiments in 24-well plates and growth arrested for 72 hours with 0.1% calf serum/DMEM. Cells will be released from Go by the addition of 5% calf serum/DMEM or with equal volumes of 10% calf serum and ECCM with or without various amounts of protamine sulfate. In addition, similar amounts of medium with and without ECCM and protamine sulfate will be added to 24-well plates devoid of SMCs to determine if formation of the complexes is cell-dependent. After one, two, and three days, the plates will be observed and photographs taken of the cells to determine if complexes are present and if they change conformations over time, as heparin and protamine complexes did in our preliminary experiments.
Determine if protamine and ECCM affect SMC growth in the same manner as protamine and heparin: The SMCs plated for the above experiment will be washed with Ca++- and Mg++-free PBS and removed from the wells by trypsinization. Cell number will then be determined using a Coulter counter.
Expected Results:
The results from these experiments will be compared with the results from the heparin and protamine studies to ascertain if heparan sulfate reacts with protamine as heparin does. If so, this indicates that studies of protamine and heparin interactions may apply to the way in which protamine reacts with heparan sulfate present in the body when it is used clinically. If the results are different, this may suggest that protamine's effect in clinical settings when used to reverse systemic heparin administration and as part of insulin preparations should be examined separately, using heparin and heparan sulfate in different experiments.
Summary
In this proposal we have outlined experiments aimed at further identifying the way in which protamine and heparin interact and how they affect aortic SMCs. We plan to examine the way in which the protamine/heparin complexes change over time, and if the effect of these complexes on SMCs is time-dependent. This information may allow us to further understand how protamine affects arterial tissue after reversing systemic heparin administration. Finally, we intend to test if protamine interacts with heparan sulfate in the same way it interacts with heparin, and if the effects on SMC growth are similar with protamine and heparan sulfate or heparin. This will allow us to determine if different experiments should be preformed to study the clinical implications of using protamine-insulins and administering protamine to reverse heparin's anticoagulative effect.
Acknoledgements
This work was conducted in the laboratory of Dr. Elazer Edelman. I would like to thank him for his guidance. I also would like to thank Elle Nugent and David Ettenson for their technical assistance and guidance.
References
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