Ambient oxygen tension modulates endothelial fibrinolysis

Ambient oxygen tension modulates endothelial fibrinolysis Jonathan P. GerBer, MD, Leland Perry, BS, Gilbert L'Italien, BS, N a n c y C h u n g - W e l c h , P h D , Richard P. Cambria, MD, Roslyn Orkin, PhD,

and William M. Abbott, MD, Boston, Mass. Purpose: Vascular procedures reoxygenate ischemic endothelial cells (EC) and arterialize saphenous vein (HSV) EC. The balance between the EC-derived fibrinolytic components, plasminogen activator (tPA), and plasminogen inhibitor (PAI-1) contributes to maintaining thromboresistance. This balance also affects proteolysis through plasmin generation, mediating matrix metabolism endothelial migration, angiogenesis, and theoretically affecting the development of intimal hyperplasia. Methods: To explore the impact of varying oxygen tensions on EC fibrinolysis, HSV and human umbilical vein (HUV) were subjected to POe of 40 mm Hg for 24 hours with restoration of P O e t o 150 mill Hg for 24 hours. The tPA and PM-1 antigen and tPA/PAI-1 antigen ratio in conditioned media (CM), expressed as ~ or $ % change, normalized for cell count, versus controls, were analyzed by enzyme-linked immunosorbent assay. Cellular tPA and PAI-1 mKNAs were assessed by Northern analysis. Results: The tPA but not PAI-1 was significantly decreased after the first 24 hours in HSVEC and significantly decreased after 48 hours in both HUVEC and HSVEC when compared with controls. Messenger KNA for tPA was unchanged but PAI-1 mRNA increased significantly for HSVEC and HUVEC after 24 hours of Po 2 of 40 mm Hg, returning to baseline within 24 hours of P O e t o 150 mm Hg restoration. Conclusions: These data support the hypothesis of a fibrinolytic shift after altered ambient O 2 tensions exposure in endothelium and demonstrate that HSVEC are more sensitive to altered O 2 tension than HUVEC. Altered 0 2 tensions depress EC fibrinolysis in this model. (J VASC SURG 1993;18:93%46.)

Long-term complications affecting the patency of vascular reconstructions are usually due to recurrent or progressive atheromatous disease, late stenosis of autogenous grafts, or problems at the graft/native artery interfaces, such as intimal hyperplasia) Vascular endothelial cells mediate normally occurring thrombotic, fibrinolytic, and proteolytic events and play a major role in maintaining normal vessel wall Stl-uct~i-e. 2"4

The organizing thesis of our investigation is that revascularization of limbs and organs exposes e n d o thelium of both autogenous grafts and native arteries From the Division of Vascular Surgery, Massachusetts General Hospital, Boston. Presented at the SeventhAnnualMeetingof the Eastern Vascular Society,Philadelphia, Pa., April 29-May2, 1993. Reprint requests: JonathanP. Gertler,MD, MassachusettsGeneral Hospital, Division of Vascular Surgery, 15 Parkman St., Suite 464, Boston,MA 02114. Copyright © 1993 by The Society for Vascular Surgery and International Societyfor CardiovascularSurgery,North American Chapter. 0741-5214/93/$1.00 + .10 24/6/51149

to oxygen tensions differing from the level to which they were exposed before revascularization. This alteration in oxygen tension theoretically might lead to altered fibrinolytic functioning of the endotheHum, which, in turn, could impact on vascular wall homeostasis. It is unclear to what degree venous endothelium, normally existing at low Po2s , shifts fibrinolytic phenotype when exposed to higher 0 2 tensions or whether ischemic arterial endothelium exhibits analogous behavior at lower 0 2 tensions. It is also unclear whether after prolonged periods at lower 02 tensions, cells constitutively adapt but again change function after introduction of normal 0 2 tension into their environment. These questions are germane to the behavior of arterialized venous grafts and revascularized arteries in previously ischenfic organs and limbs. In chronic occlusive problems there is limitation of blood flow and oxygen delivery to downstream vessels of the affected organ or limb. Revascularization of chronically ischemic limbs and organs may result in reperfused, previously chronically ischemic 939

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arterial endothelium and also in arterialization of venous endothelium when an autogenous graft is used. Because endothelium is capable of producing and binding multiple factors that are responsible for vessel wall metabolism and structure, characterizing the endothelial response to altered oxygen tensions remains critical to understanding the frequently observed long-term complications associated with the vascular surgical interventions described above. Although previously regarded as primarily involved in thromboregulation, increasing attention is being given to the plasminogen activator system's regulation of long-term cellular interactions with the surrounding matrix) Oxygen in the cell's environment is now recognized as a critical regulator of myriad cellular responses and not just a nutrient that causes global toxicity when critically low levels are reached.6 To begin to explore the interactions among oxygen, the plasminogen activator system, and the endothelial matrix, the antigenic and message expression for both tissue type plasminogen activator (if'A) and plasminogen inhibitor type I (PAI-1) in endothelial cells maintained at differing O 2 levels were investigated. The experiments were also designed to explore the impact of varying oxygen tension on endothelial cell-based fibrinolysis in endothelial cells of different origin; that is, human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC). MATERIAL AND METHODS Culture and assay protocols. HUVEC and HSVEC were cultured according to previously reported techniques .7 In brief, veins were transported immediately in sterile M199 medium (GIBCO Laboratories, Life Technologies, Inc., Gaithersburg, Md.) and cannulated at the distal end. Warm collagenase solution (0.1%) was introduced with gentle distension. After incubation at 37 ° C for 10 minutes, luminal contents with an additional 5 ml M199 were centrifuged at 1000 RPM for 10 minutes at 10 ° C. The pellet was resuspended in complete media with 5 gm/L glucose and plated on a gelatin-coated T25 flask. At confluence, cells were removed with trypsin and plated onto T75 flasks. Cells were identified as endothelial in origin by uptake of low-density lipoprotein and were used up to passage 4. Experimental design. HUVEC and HSVEC were subjected to POz of 40 mm Hg for 24 hours with a previously described method for inducing lower oxygen tension in endothelial cells.7 After 24 hours of lower ambient oxygen, cells were exposed to 24 hours of Po 2 of 150 mm Hg. Simultaneous

JOURNAL OF VASCULAR SURGERY December 1993

controls of the same cellular origin were maintained a t P O 2 of 150 mm Hg for 48 hours. Endothelial cell viability at the end of the experimental or control periods was assessed by trypan blue exclusion. Hemocytometer counting by an individual blinded to the growth conditions of the cells was used. Phase contrast microscopic views of the monolayers were taken at each time point to record any morphologic variations, during the experimental procedures. PO2 level of media was verified by use of a standard blood gas analyzer, and pH was normalized as needed with sodium bicarbonate in both control and experimental groups to maintain pH of 7.4. Media was replaced every 24 hours and frozen at - 70 ° C for later analysis of tPA and PAI-1 antigen and tPA/PAI-1 antigen ratio in conditioned media (CM), expressed as increased or decreased percent change, normalized for cell count versus controls. Cellular mRNA was isolated from both 24-hour and 48-hour experimental and control cells for assessment by Northern analysis. Five experiments were executed for each time point. tPA antigen assay. An enzyme-linked immunosorbent assay was used for tPA antigen. Two hundred microliters CM was incubated for 2 hours at room temperature on a microtiter plate precoated with mouse monoclonal anti-tPA. After incubation, five successive washings were carried out, and anti-tPA peroxidase conjugate (mouse monoclonal anti-tPA antibody coupled with peroxidase) was added with an additional 2-hour room temperature incubation. The plates were rewashed, and 200 ~1 of orthophenylenediarnine substrate was added for 6 minutes followed by 100 ~11 mol/L HCI for an additional 10 minutes. Optical density at 492 nmol/L was measured. Unknown samples were quantitated on the basis of a standard curve with reference tPA purified from human origins. PAI-1 antigen determination. A similar enzyme-linked immunosorbent assay was used for PAI-1. Two hundred microliters CM was added to microtiter plates coated with mouse monoclonal anti-PAI-1 F (ab)2 fragments and incubated for 1 hour at 18 ° to 25 ° C. Thorough washing with isotonic buffer was followed by the addition of anti-PAI-1 peroxidase conjugate (mouse monoclonal anti-human PAI-1 antibody coupled with peroxidase) and incubated for 1 hour at 18 ° to 25 ° C. After repeat washing, orthophenylenediamine substrate (200 ~l) was added at 18 ° to 25 ° C for 3 minutes, and 3 mol/L H2SO 4 (50 ~1) was introduced for 10 minutes. Optical density was measured at 492 nm, and results were compared with a standard curve generated with PAI- 1 reference material on a log-log curve.

JOURNAL OF VASCULAR SURGERY Volume 18, Number 6

R N A extraction and northern hybridization. Total cellular RNA was extracted from cell monolayers as described previously,s Briefly, cells were removed with trypsin and pelleted. Cell pellets were homogenized in 4 mol/L guanidinium thiocyanate containing 0.5% sodium N-lauryl sarcosinate. RNA was concentrated through a 5.6 mol/L cesium chloride cushion at 32,000 rpm for 16 hours on a Beckman SW-41 ultracentrifuge (Beckman Instruments, Inc., Brea, Calif.). The pellet was washed in 70% ethanol, dryed, and stored in diethyl pyrocarbonate-treated water at - 7 0 ° C until it was used. PBR 322- and PUC-based plasmids for PAI-1 and tPA were used. Two hundred microliters of Escherichia coli (DH5) competent cells and 0.1 mg of plasmid DNA were mixed, incubated at 0 ° C for 30 minutes, and heat shocked at 37 ° C for 5 minutes. One milliliter luria broth medium was added, and cells were grown in a rotary drum for 45 minutes at 37 ° C. Cells were plated on luria broth/agar/ampicillin plates and grown with a yield of 106 colonies/ng DNA inserted. Bacterial pellets were obtained by centrifugation of 500 ml culture at 4000 rpm for 15 minutes. Supernatant was discarded, and the pellet was resuspended in 10 ml of 50 mmol/L glucose. Twenty-five millimolars per liter Tris-CL (pH 8.0) 10 mmol/L ethylenediamine tetraacetic acid (EDTA) 1 ml of lysozyme (10 mg/ml in 10 mmol/L Tris-HCl, pH 8.0) was added, followed by 20 ml 0.2 N NaOH, 1% sodium dodecyl sulfate. Gentle mixing for 10 minutes was followed by addition of 15 ml ice-cold 60 ~1 5 mol/L Kacetate, 11.5 ml glacial acetic acid, 289.5 /~1 H 2 0 , followed by additional mixing and storage at 0 ° C for 10 minutes. The lysate was centrifuged at 4000 rpm for 15 minutes at 4 ° C, and the supernatant was transferred and filtered through cheesecloth. A volume of 0.6 isopropanol was added for 10 minutes, and the mixture was centrifuged at 5000 rpm for 15 minutes. The supernatant was discarded, and the pellet was rinsed with 70% ethanol, which was drained and evaporated. The final pellet was dissolved in Tris-EDTA pH 8.0. For preparative electrophoresis oftPA and PM-1 CDNA fragments, 40 ~tl of 10x high-salt buffer, 40 ~zlofmaxi prep DNA, 40 ~1 of ECOR1, and H 2 0 to a total volume of 400 ~1 was incubated for i hour at 37 ° C. A GTG agarose gel (Sigma Chemical Co., St. Louis, Mo.) (200 ~1, 20 × 20 cm), was prepared with 0.5% agarose with 1 Kb ladder (BRL) used as a size marker. The gel was run overnight at 40 V in 1 × TBE(50 mmol/L Tris) without ethidium bromide followed by 15 minutes light staining with ethidium bromide. The desired band was excised on

Gertler et al.

941

an ultraviolet illuminator and placed in a dialysis bag containing 400 ~1 0.2 x TBE buffer. The bag was placed in a horizontal agarose gel box with 0.2 × TBE buffer and electroeluted at 300 V for 1 to 3 hours. The solution was mixed and removed and placed in a 1.5 ml microfuge tube with an additional 100 txl 0.2 × TBE buffer (total 500 ~1) used to rinse the bag. The solution was phenol/chloroform extracted and ethanol precipitated. The precipitate was dissolved in 50 to 100 ~t of Tris-EDTA 10/1, ph 0.8, and quantitated on a test gel with lambda DNA digested with Hind III with a final concentration adjusted to 25 ng/ml. For Northern hybridization, 10 ~tg RNA from each T75 flask were loaded on 1.5% 3-(Nmorpholino) propanesulfonic acid-formaldehyde agarose gels and electrophoresed at 5 V/cm. After overnight capillary transfer to uncharged nylon membranes in 25 mmol/L sodium phosphate (ph 6.5), the RNAs were fixed by ultraviolet irradiation. The membranes were prehybridized for 1 to 2 hours at 65 ° C in 50 mmol/L Tris-HCE per 0.1%. Napyrophosphate, 1 mol/L NaCI per 0.2% Ficoll (Pharmacia, Biosystems, Piscataway, N.J.) per 0.2% BSA per 1% SDS 0.1 mg/ml mRNA, then hybridized overnight with the labeled probe(s) (labeled by random primer extension). High stringency wash was 75 mmol/L NaC1 per 7.5 mmol/L Na citrate/0.5% sodium dodecyl sulfate at 65 ° C. Filters were air dried and exposed to Kodak XAR film (Eastman Kodak Co., Rochester, N.Y.) with intensifying screen at 7 ° C. Quantitation was achieved by densitometric analysis. Statistical significance for all assays was determined by paired Student's t test. RESULTS Ceil'viability as assessed by trypan blue exclusion exceeded 93% in all groups with no differences noted among low- and high-oxygen groups. Phase contrast microscopy revealed no differences in alignment of cells or cellular configuration among the different groups. POE for the hypoxic group were confirmed at 40 mm Hg after hypoxic incubation with controls and reoxygenated cell media maintained at Po E of 150 mm Hg throughout the appropriate time COUrSeS.

The percent changes versus controls for PM-1 and tPA antigens and PM-1/tPA antigen ratios for HSVEC and HUVEC in the first and second 24-hoUr periods are demonstrated in Tables I and II, respectively. In essence, saphenous vein endothelial cells demonstrated a significant and persistent decrease in tPA antigen, whereas a trend to increasing PAL 1 was noted consistent with an antifibrinolvtic

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942 Gertler et al.

December1993

Table I. Percent change vs control tPA and PAI-1 HUVEC

First 24 hours, Po 2 = 40 m m H g Second 24 hours, P O 2

=

No.

tPA

PAI-1

tPA/PAI-1 ratio

5

$ 54.6 -+ 6.9*

$ 4.6 + 5.5

$ 59.2 + 2.6*

5

$ 51.2 + 5.3*

1' 15.4 + 8.4

$ 50.4 --+ 1.8"

PAI-I

tPA/PAI-1 ratio

150 mm Hg *Less than 0.01, paired t test. Table II. Percent change vs control tPA and PAI-1 HSVEC

First 24 hours, Po 2 =

No.

tPA

5

$ 1 1 . 0 + 6.8*

~ 4.5 + 2.7

$ 1 5 . 0 + 11.8

5

$ 48.0 -+ 5.9"

i' 16.0 + 9.8

$ 51.2 + 4.3*

40 mm Hg Second 24 hours, Po 2 = 150 rnm H g *Less than 0.01, paired t test.

shift. The tPA/PAI-1 ratio was statistically depressed in both 24-hour periods. HUVEC demonstrated greater initial resistance to hypoxia; however, a preliminary decrease in tPA antigen during the first 24 hours was followed by a statistically significant further decline in tPA antigen presence in CM. PAL1 was unaffected in HUVEC; however, tPA/PAI-1 ratios of course reflected decreased tPA and were also consistent with a shift away from fibrinolytic function. Northern blots for tPA and PAI-1 (both 3.4 and 2.5 Kb species) are depicted in Figs. 1 and 2. The mRNA for PAl-1 increased significantly for HSVEC and HUVEC after 24 hours of P O 2 of 40 mm Hg, returning to baseline within 24 hours of higher oxygen restoration. The tI'A mRNA was unaffected in this model for the time periods studied. DISCUSSION The data presented in this report support the hypothesis of altered fibrinolytic activity after changes in ambient oxygenation, with responses apparent at both transcriptional and translational levels. HSVEC are more sensitive to altered oxygen tension than are HUVEC, which is not surprising given the generalized ability of fetal tissue to resist hypoxia and hypoxemia. Both cell types clearly respond in this model and demonstrate a sensitivity to ambient oxygen. It is suggested by our data that lowered ambient O 2 tension affects tPA antigens without mRNA effect and PAI-1 mRNA without antigen effect. Possibilities for these discrepancies include altered binding or release characteristics in addition to signaling mechanisms at the nuclear level. As the message level affect for PAI-1, which was noted in our data was not reflected in protein

production, it appears that altered ambient 02 tension renders its effects on both transcriptional events and existing stores of protein. The tPA, which is bound to cell surface and is present intracellularly, diminished in the CM, possibly reflecting increased binding or decreased release at the apical surface of the cell. PAI-1 mRNA rapidly regressed to normal levels in the second 24 hours of experimental conditions. It is possible that the 24-hour increase in PAL1 mRNA reflected an earlier increase with normalization still resulting in enhanced levels at the 24-hour point. This will be further investigated by nuclear runoff studies. The reversibility that is demonstrated in these experiments, the functional significance of transcriptional changes, and the persistence of this response after longer periods of altered oxygen tension remain critical questions to be answered. The tPA and PAI ratios, as used for analysis in this and other reports, 9 have been related to angiogenesis and endothelial cell migration in vitro. Transforming growth factor-J3 (TGF-[3), which shifts the tPA/PAI-1 ratio against proteolysis, inhibits tubelike structures produced in vitro by endothelium exposed to basic fibroblast growth factor (B-FGF).9 Plasmin, released by tPA from plasminogen, can activate TGF-[3, release complexes of B-FGF/glycosaminoglycan from cell surfaces and matrixes, and, through focused proteolysis, activate a variety of other growth factors as wellfi,I° Thus the plasminogen activator system likely plays a major role in vascular wall remodeling, which may be a critical aspect of clinical vascular events such as intimal hyperplasia, which in late stages is a highly fibrotic lesion. The stimulus for altered tPA-PAI-1 activity alteration after revascularization remains unknown.

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Gertler et al.

943

z A

o

u

.I~

~,

2:

:,,

2:

ua kb ....

.......... 7 . ~ .........

4.~

.....

2.37

.,,

0.24

~

Fig. 1. tPA Northern blot for HSVEC and HUVEC (experimental and control). Investigations are increasingly emphasizing plasminogen activator activity directionality, which is not addressed in this report. ~ It may be that the cells' response as seen in CM does not reflect the functional response on a subcellular level. This concept of "focused proteolysis" may entail a concentration of activity at the abluminal or luminal surface, the focal contact protein level or the cytoskeletal level. Our current efforts are addressing this directionality, investigating both luminal and abluminal of secretion and subcellular plasminogen activator activity. The endothelial cell responds to lowered ambient oxygen tensions in a variety of ways. Oxygen tension is a signal sensed and transduced by the endothelium and not merely a cell toxic event with attendant release of mitogenic or other products. New protein synthesis contributes to preserved endothelial viability in the setting of hypoxic stress, ~2 suggesting that the endothelium has a mechanism for responding to differences in ambient oxygen tension. Although the endotheliurn is capable of producing oxygen free radicals and certain products, such as endothelium-

derived relaxing factor, are probably released as a result of free radical activity,13 the increased production of endothelin-1 ~4 and a reversible increase in transcriptional rate for TGF-13 mRNA after hypoxia 14 indicate endothelial ability to sense oxygen tension without altered viability and with upregulation of specific genes. The data presented in this report further support this concept demonstrating that both transcriptional and translation events in endothelial cells occur in response to altered ambient oxygen. The time points selected in this experiment are representative of a longer time exposure to lower oxygen tension than is usually seen in the setting of acute vascular injury. This model is being expanded to include longer time periods more reflective of cells existing chronically at lower 0 2 levels. The central concept behind these investigations is related to the interaction noted between the plasminogen activator system and angiogenesis as noted above. A link between regulation of matrix degradation and oxygen tension in endothelial cells is possible; however,

944

JOURNAL OF VASCULARSURGERY December 1993

Gertler et al.

¢0 ua

~

-I-

,,..,,

kb 9.49 7.46 4.40 2.37 1.35

0.24

Fig. 2. PAI-1 Northern blot for HSVEC and HUVEC, PAI 3 designated PAI-1 probe (experimental and control). the data presented in this report cannot be used for anything but speculation in this regard. Oxygen changes dearly affect fibrinolysis and thus may impact on proteolysis through plasminogen activation and inhibition. The mechanisms by which 02 affects cells are not clear; however, free radical formation in models of anoxia and reoxygenation has been noted to alter fibrinolysis,16 and adenosine mediation has also been observed in some responses of cells to hypoxia.~7 Others have noted new protein synthesis after hypoxic stress in endothelium, which differs from heat shock protein production, but a signal mechanism for this oxygen effect has not been determined. 18 The end-stage lesions of intimal hyperplasia are hypocellular and laden with excessive fibrotic (matrix) elements. A shift in oxygen tension in arterialized venous cells theoretically could alter the balance of fibrinolysis. This in turn affects the activation of plasmin and results in suppressed proteolysis and thus excessive accumulation of matrix molecules

contributing to the lesions of intimal hyperplasia. Exploration of these phenomena for differing time periods, their reversibility, their location(s) in the cell, and their impact on growth and behavior of endothelial cells with respect to matrix production have not been explored to date in a model simulating vascular grafting and remains the focus of our work. Data are presented that support the hypothesis of a fibrinolytic shift after altered ambient 02 tensions exposure in endothelium, regulated at both gene and protein level. Altered 02 tensions depress endothelial cell fibrinolysis in this model in both HUVEC and HSVEC, although HSVEC are more sensitive to the experimental conditions exposed. REFERENCES

I. Licalzi LK, Stansel HC. Failure of autogenous reversed saphenous vein femoropopliteal grafting, pathophysiology and prevention. Surgery 1987;91:352-7. 2. Erickson LA, SchleffRR, NyT. The fibrinolyticsystem of the vascular wall. Clin Hematol 1985;14:513-30. 3. Rodgers GM, Greensberg CS, Shuman MA. Characterization

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of the effects of cultured vascular cells on the activation of blood coagulation. Blood 1983;61:1153-62. 4. Ross R. The pathogenesis of atherosclerosis: an update. N Engl }"Med 1986;314:488-500. 5. Vassali JD, Sappino AP, Belini D. The plasminogen activator/plasmin system. J Clin Invest 1991;88:1067-72. 6. Adair TH, Gay WJ, Montani JP. Growth regulation of the vascular system: evidence for a metabolic hypothesis. Am J Physiol 1990;259(3Pt2) :R393-404. 7. Gertler JP, Weibe DA, Ocasio V, Abbott WM. Hypoxia induces procoagulant activity in human venous endothelium in vitro. J VASCSVRG 1991;13:428-33. 8. Chirgwin JM, Przybyla AE, Mac Donald RJ, Rutter WJ. Isolation of biologically active ribonucleotides from sources enriched ribonuclease. Biochemistry 1979;18:5294-9. 9. Pepper MS, Belini D, Vassali JP. Transforming growth factor-beta 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro. J Cell Biol 1990;111:743-55. 10. Rifldn DB, Mostcatelli D, Bizif J, et al. Growth factor control of exttacellular proteolysis. Cell Diff Dev 1989;32: 313-8. 11. Hakkert BC, Rentenaar JM, Van Mourik JA. Monocytes enhance the bidirectional release of type I plasminogen activator inhibitor by endothelial cells. Blood 1990;76:227278.

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12. Zimmerman LM, Levine RA, Farber HW. Hypoxia induces a specific set of stress proteins in cultured endothelial cells. J Clin Invest 1991;87:908-14. 13. Gryglewski RJ, Palme RM[I,Moncada S. Nitric oxide release accounts for the biological activity of endothelium derived relaxing factor. Nature 1986;31:385-94. 14. Gertler JI', Ocasio VH. Endothelin production by hypoxic htmaan endothelium. J VAsc SURG1993;18:178-84. 15. Santilli SM, Fiegel VD, Aldridge DE. Rabbit aortic endothelial cellhypoxia induces secretion of transforming growth beta and augments macrophage adhesion in vitro. Ann Vasc Surg 1991;5:429-38. 16. Shatos MA, Doherty JM, Offer T, et al. Modulation of the fibfinolytic response of cultured human vascular endothelium by extracellularity generated oxygen radicals. J Biol Chem 1992;267:597-601. 17. Shryock JC, Rubio R, Berne RM. Release of adenosine from pig aortic endothelial cells during hypoxia and metabolic inhibition. Am J Physiol 1988;254:H223-H229. 18. Zimmerman LH, Levine RA, Farber HW. Hypoxia induces a specific set of stress proteins in cultured endothelial cells. J Clin Invest 1991;87:908-14. Submitted May 11, 1993; accepted Aug. 25, 1993.

DISCUSSION

Dr. Thomas F. Panetta. The concept proposed by the authors is that endothelial cells subjected to decreased oxygen tension demonstrate procoagulant activity manifest by a decreased production of tPA. Dr. Gertler and his colleagues have developed a unique in vitro model of vascular ischemia with appealing potential applications. However, some questions with regard to the ischemic setting of endothelial cells come to mind. Can you correlate the oxygen tension in your media at a Po 2 of 40 mm H g with that of venous blood from an ischemic extremity? Keep in mind that in the absence of hemoglobin, maximum free oxygen concentrations in serum is extremely low, even with the use of hyperbaric oxygen. Therefore a Po 2 of 40 mm H g in medium may not represent a hypoxic setting for venous endothelium. Did the authors use electron microscopy to demonstrate whether ultrastructural changes such as vacuolization, decreased microvilli, or mitochondrial swelling occurred to document that these endothelial cells were indeed ischemic? Would you comment on whether the results might represent a response to varying oxygen tension rather than the result of true ischemia? The second question relates to the differences between human umbilical vein and saphenous veins. The authors propose a teleologic explanation for the decreased sensitivity of umbilical vein to decreased oxygen tension. The decreased sensitivity of human umbilical veins may be even more pronounced if one considers that normal oxygen tension of umbilical veins is nearly three times that o f

saphenous vein. Do you have any supporting evidence that the responses of H U V E and HSVEC are the same? Do you have any evidence that further deterioration in H U V E C with reestablishment of oxygen is mediated by free radicals, and if so, why don't HSVEC deteriorate with oxygenation? Are HSVEC therefore less sensitive to free radical injury? The authors propose that an ischemic extremity may develop changes in arterial endothelial cells and HSVEC, for example, after arterial thrombectomy or after arterialization o f a saphenous vein graft. It seems more likely that procoagulant activity in the venous endothelium of the deep venous system subjected to the same low oxygen tensions should predispose these patients to a much higher incidence of deep vein thrombosis (DVT). Would you please comment? The leap to explain intimal hyperplasia, angiogenesis and the development of late lesions in arterialized vein grafts seems premature. However, the concept of focused proteolysis lends nicely to the concept of subendothelial migration of smooth muscle cells as proposed by Clowes and Reidy (J VAse SVRG 1991; 13: 885-91 ) rather than the migration of endothelial cells as you proposed. Would you comment? Clearly vein graft lesions are of multifocal nature and have a high correlation with both early and late graft failures and preexisting saphenous vein disease. Would you speculate on the role of increased procoagulant activity in the development of saphenous vein increased wall thickness, calcifications, and thrombosis in saphenous veins that

946

Gertler et al.

we have noted to have preexisting disease before being used as a bypass when harvested from an ischemic extremity? Dr. Jonathan P. Gertler. You're tight in pointing out that the Po 2 may not actually be ischemic, and I should emphasize that this is not a model of ischemia. There are a variety of other studies on other regulatory systems studying ischemia reperfusion where the Po 2 is actually dropped to a level of 14 mm H g or less. Ours is not a model of venous ischemia. It's more a model of routine venous tensions and reoxygenation of them as follows arterialization of a vein graft. It is not supposed to be a model of venous hypoxia. I think one would have to drop the 02 much lower for true ischemia to take place. With regard to your question on electron micrografts; no, I don't have any correlation of that. We are not trying to damage these cells by ischemia/reperfusion syndrome; we are trying to alter ambient oxygen tension over the long term in a way that the cells remain viable and fimctional; I believe that's corroborated both by functional changes seen in this work and functional changes seen in other work. This is not a model of true ischemia but one of altered oxygen tensions. True ischemia actually reflects a much lower oxygen tension. I don't have any information on HUVECs and other species. They may be more or less sensitive; I don't have the data available to analyze it. Fetal tissues overall are more resilent in tissue culture across the board. With regard to saphenous vein versus umbilical vein, saphenous vein is much more fastidious, much more difficult to grow, much harder to transport, and requires the presence of growth factors. I think one of the difficulties in this work is that HUVECs don't require growth factors, and yet growth factors have a pronounced effect on expression of plasminogen activators. If we were to compare saphenous vein grown in growth factors with the standard H U V E C preparation without growth factors, we would be undermining the data terribly. As it is, it's difficult. The growth conditions for cells vary so widely among different species and cell types that interpretation becomes difficult. Endothelin-I was produced in this model in an augmented fashion. When free radicals were inhibited, the augmentation was even more pronotmced. Free radicals seemed to have nothing to do with signaling for endothelin-I production or might have inhibited the ongoing synthesis or release of endothelin-I somewhat by damaging cellular machinery; however, the presence of free radicals was unrelated to the response of cells to hypoxia. Free radicals are known to be involved in the elaboration of endothelium-derived relaxing factor after lower ambient oxygen tensions but that doesn't seem to be relevant to these findings. Finally, with regard to DVT, I think you actually answer that question yourself when you said that this is not ischemic endothelium. SO I don't think that that will make these patients more prone to DVT. In addition, the plasminogen activator system, although it's critical in initial

JOURNALOF VASCULARSURGERY December 1993

thromboregulation and thromboresistance, does have directionality. What we're seeing here grossly in CM and m R N A needs to be focused within the cell to see whether this is a luminal or an abluminal effect. With regard to saphenous vein grafts, there is actually good evidence that the valve pockets are much more hypoxic than the rest of the cell. Those have actually been measured in a 1973 article in the BritishJournal of Surgery. The Po z in the valve pockets in that study was much less than the venous blood itself. That was believed to be one of the initial causes of DVT. It may well be that a different oxygen environment is partly responsible. Lastly, with regard to Clowes' work, yes, certainly smooth muscle cell migration is critically important in the whole pathophysiologic condition of intlmal hyperplasia and proteolysis, which is mediated by the endothelial layer may be critically important in that as well. Dr. Michael Golden (Philadelphia, Pa.). Given the fact that the PAI-1 and tPA are not the sole members of the proteolytic family or the plasminogen activator family and inhibitor family. Have you looked at any of the other members like urinary type plasminogen activator, which you have mentioned you will be looking at and the other plasminogen activator inhibitors? In addition, the various tissue proteases that are not actually specifically members of the plasminogen activator family, and also the role of potential growth factor elaboration by the hypoxic cells, since there is excellent evidence that the ischemic, or shall we say, hypoxic endothelium does elaborate elevated levels of growth factors and they can also feed back on the plasminogen activator system. Dr. Gertler. Intimal hyperphsia, if you want to use that as the clinical endpoint of a study like this, is obviously multifactorial, and we have ongoing projects looking at a combination of biomechanical forces, hypoxia, biologic response and that has to include various growth factors. It's very difficult to limit or to choose the ones to study, because there are so many of them and their interactions are complex. With respect to the other proteases that are present, we are not going to study these specifically through enzymatic analysis, but we are looking at collagen expression, deposition, secretion in the cells. We hope to at least address the question of how hypoxia impacts on the end process, which is really the formation of the fibrotic end stage lesion, so I think all of these points are very valid and need to be continued. Dr. Frank J. Veith (Bronx, N.Y.). Have you thought of comparing your results in these low-oxygen situations with the results of endothelial cells derived from arteries? Can that be studied? Dr. Gertler. It can be studied, but it's more difficult because the cells are more fastidious. We have cadaveric and transplant samples in the laboratory. Hyperoxia, which might have implications as well, is probably the other end of it that's critically important. It doesn't seem to be the critically low level, it seems to be that it's a deviation from the norm that signals the cell.