Role of Prostacyclin in the Cardiovascular Response to Thromboxane A2 Yan Cheng, Sandra C. Austin, Bianca Rocca, Beverly H. Koller, Thomas M. Coffman, Tilo Grosser, John A. Lawson, and Garret A. FitzGerald

Supplementary Material

Supplemental Figure 1. Examples of the response to vascular injury in uninjured and injured IPWT and IPKO mouse carotid arteries. (A) Uninjured artery in IPWTs, (D) injured IPWTs, and (G) injured IPKOs are representative segments from hematoxylin and eosin (H&E)-stained sections; and (B) uninjured artery in IPWTs, (E) injured IPWTs, and (H) injured IPKOs are stained with van Gieson dye. BrdU staining for cellular proliferation in (C) uninjured artery in IPWTs, (F) injured IPWTs, and (I) injured IPKOs. The arrows and the arrowheads indicate the internal elastic laminae and the external elastic laminae, respectively. Scale bar, 50 m.

Medium version | Full size version

---

Supplemental Figure 2. Both the hyperplastic response to injury and the resultant platelet activation, as reflected by urinary Tx-M, are reduced in TPKOs. (A) Injured TPWTs and (D) injured TPKOs are representative segments from H&E-stained sections; (B) injured artery in TPWTs and (E) injured TPKOs are van Gieson-stained; (C) injured artery in TPWTs and (F) injured TPKOs are stained with BrdU. The arrows and the arrowheads indicate the internal elastic laminae and the external elastic laminae, respectively. Scale bar, 50 m

Medium version | Full size version

---

Supplemental Table 1. Morphometric analysis of carotid arteries and Tx-M biosynthesis after vascular injury in KO mice and their corresponding WT littermates. Results are means ± SEM of values from eight to nine animals in each group. Statistical analysis:*P < 0.05,**P < 0.01 compared with corresponding WTs .
	IP		TP		IPTP
	WT	KO	WT	KO	WT	KO
Intimal area	20.2 ± 3.8	51.6 ± 13.0**	32.3 ± 7.3	12.9 ± 3.6*	30.7 ± 15.0	19.7 ± 4.1
Intimal to medial area	0.5 ± 0.1	1.0 ± 0.1**	0.7 ± 0.1	0.3 ± 0.1*	0.66 ± 0.14	0.66 ± 0.11
Percent lumen stenosis	29.9 ± 5.8	58.1 ± 7.8*	49.9 ± 4.6	27.3 ± 4.7**	34.5 ± 5.7	42.5 ± 6.9
Proliferative cells (cells/cross section)	111.1 ± 13.7	188.5 ± 26.9*	121.4 ± 18.3	61.5 ± 18.2*	101.0 ± 11.3	123.7 ± 17.7
Urinary 2,3-dinor-TxB2 (fold over basal after injury)	2.0 ± 0.4	5.4 ± 1.1*	2.8 ± 0.2	1.4 ± 0.2**	2.1 ± 0.4	2.3 ± 0.4

---

Methods

Mice. In all cases, transgenic mice deficient in the indicated gene were compared with appropriate strain-, age-, and sex-matched control animals.

The IPKO mice were generated by homologous recombination in embryonic stem (ES) cells and maintained on a mixed 129 × C57BL/6 genetic background. IP^-/- and control littermates were identified in litters generated by the intercross of IP^+/- animals by polymerase chain reaction analysis of genomic DNA isolated from tail biopsy samples. TPOE mice were generated and maintained on the B6SJLF₁/J genetic background. Mice carrying the transgene and wild-type littermate controls were identified by Southern blot analysis as described previously (1). TPKO mice were generated as described previously (2) and backcrossed for six generations onto the BALB/cJ genetic background. Heterozygous animals were intercrossed, and the litters were screened by Southern blot analysis to identify both TP^-/- and wild-type controls. TPIP DKO mice were generated by intercrossing 129 TPKO and 129 IPKO mice. These 129 congenic lines were generated by breeding the chimeric animals generated from the TP and IP mutant ES cells directly with 129 females. The investigator was unaware of the genotype throughout the experiment.

Experimental protocol. All procedures were approved and animal husbandry was overseen by the Institutional Animal Care and Usage Committee of the University of Pennsylvania. The left carotid artery was injured, using a modification of a described procedure (3, 4). Both KO and WT male mice (24 to 28 weeks old, 30 to 35 g) were maintained on a standard diet and water ad libitum. Mice were anesthetized on day 0 using ketamine (Webster Veterinary Supply Inc., Sterling, MA) 100 mg per kg body weight given intraperitoneally and acepromazine (Fermenta Animal Health Co., Kansas City, MO) 5 mg/kg given intraperitoneally. The left carotid artery was surgically exposed with aseptic techniques and isolated from the surrounding tissues. The proximal common and internal carotid arteries were occluded with microvascular clamps, and the external carotid artery was identified and ligated with 5-0 silk. A curved, flexible angioplasty wire (0.35-mm diameter; Cook Group Inc., Bloomington, IN) was introduced into the external carotid artery and advanced to the left common carotid artery, and was passed along the vessel four times, with rotation. Subsequently, a catheter (0.54-mm diameter; Cole-Parmer Instrument Inc, Vernon Hills, IL) was immediately introduced into the common carotid artery, passed four times, with rotation, and then removed. The external carotid artery was tied off. The clamps were removed to reestablish normal anterograde flow, and the incision closed with sutures. The right carotid artery was sham-operated and used as a control. Arteries from a second group of animals were subjected to 5% Evan's blue staining to verify that the injury created was uniform and complete, with endothelial denudation of the injured artery consistently exceeding 95% of the total area. Alzet osmotic minipumps (model 2002, Alza Co., Palo Alto, CA) were placed subcutaneously via a midback incision and loaded with bromodeoxyuridine (BrdU, Sigma Chemical Co.) to deliver 25 mg per kg per day. All animals survived until the time of planned death without bleeding or infection.

Tissue collection and histology. Fourteen days after injury, animals were anesthetized and perfused with 0.9% NaCl by placement of a 22-gauge needle in the left ventricle. The mice were subsequently perfusion-fixed in situ by infusion with 4% buffered formalin (pH 7.0) at a constant pressure of 100 mmHg. Both right and left carotid arteries were harvested. Each artery was embedded in paraffin, cross-sections were made from the branch point of the external carotid and four 5-m-thick sections, 300 m apart, were processed for routine H&E staining. Adjacent sections were processed for elastin staining and immunohistochemical labeling for BrdU.

BrdU. Slides were deparaffinized with xylene by incubation for 20 min twice and then dehydrated with a series of graded ethanol solutions (70 to 100%) each for 5 min, placed for elastin staining in a solution containing alcoholic hematoxylin, 10% aqueous ferric chloride and iodine for 12 min, rinsed in distilled water, and then placed into 5% aqueous ferric chloride solution for 1 min. Sections were rinsed in tap water and then in 95% alcohol to remove the iodine. The slides were then placed into van Giesen's solution (Eng Scientific Inc., Clifton, NJ) for 2 min, rinsed in 95% alcohol, dehydrated, and examined under a cover slip.

Sections for immunohistochemistry were deparaffinized and then were washed in distilled water for 5 min twice and incubated with 2N HCl at 37°C for 15 min. After washing with PBS, the slides were further incubated with 0.05% trypsin at 37°C for 20 min. Endogenous peroxidase activity was blocked by incubation with 1% hydrogen peroxide in PBS. Slides were then washed three times in PBS. Nonspecific binding sites were blocked by incubation for 30 min with 10% serum in PBS containing Tween 20. The slides were again washed in PBS and incubated with the mouse monoclonal antibody (clone BMC 9318, IgG1; Boehringer Mannheim Co, Indianapolis, IN) for 30 min at 37°C in a humid atmosphere. Finally, the slides were incubated with anti-mouse Ig-alkaline phosphatase for 30 min at 37°C and the reaction was visualized with nitroblue tetrazolium and X-phosphate.

Morphometric analyses were performed on the H&E-stained tissue. Briefly, 10 cross sections each, from both the injured and uninjured arteries were viewed using a Nikon CCD video camera attached to a Nikon microscope (Optical Apparatus Co., Ardmore, PA) and digitized with Image Pro image analysis software (Media Cybernetics, Silver Spring, MD). A custom program (Phase 3 Imaging Systems, Glen Mills, PA) was used with Image Pro to measure the area and the perimeter of the lumen, the internal elastic lamina, and the external elastic lamina. BrdU-labeled cells within the media and intima were also counted by this custom program. The percentage stenosis was calculated as the intima area/area inside the internal elastic lamina.

TP antagonism. The TP receptor antagonist, S18886, was a gift from the Institut de Recherches Internationale Servier, Surenes, France. S18886 (1 mg, 10 mg, and 100 mg per kg body weight) or demineralized water was administered once daily by esophageal intubation, starting 2 days before injury and continuing for the next 2 weeks when the carotid artery was excised for histological staining. For the platelet aggregation assay, S18886 at 10 mg per kg body weight was administered once daily for 3 days, and on the third day, mice were anesthetized with ketamine, and blood was obtained via cardiac puncture for study of platelet aggregation ex vivo, as previously described(1).

Urinary 2,3-dinor-TxB₂ was measured by a stable isotope dilution reversed-phase (C₁₈) HPLC/MS/MS assay in which 100 ml of urine is spiked with 1.1 ng of an ¹⁸O₂-labeled analog, derivatized to the O-methoxime by addition of 40 mg methoxime hydrochloride, extracted on a C₁₈ solid-phase extraction cartridge and purified on a weak anion exchange (NH₂) SPE cartridge, before injection into the tandem quadrupole mass spectrometer (Micromass Quattro II; Micromass, Berkeley, MA). Multiple Reaction Monitoring conditions were used to generate mass to charge ratios (m/z) of 370 for the endogenous compound and m/z 374 for the internal standard. Quantification was derived from ratios of peak areas.

Statistical analysis. All values were expressed as mean ± SEM. When only two groups were compared, differences were assessed by a paired (e.g., injured versus contralateral arteries) or unpaired Student's t test (e.g., different genotypes). Multiple comparisons were first subjected to analysis of variance, before pairwise comparisons, as appropriate.

References

1. B. Rocca et al., Nature Med. 6, 219 (2000).

2 D. W. Thomas et al., J. Clin. Invest. 102, 1994 (1998).

3. W. M. Cheung, M. R. D'Andrea, P. Andrade-Gordon, B. P. Damiano, Arterioscler. Thromb. Vasc. Biol. 19, 3014 (1999).

4. K. Harada, I. Komuro, T. Sugaya, K. Murakami, Y. Yazaki, Circ. Res. 84, 179 (1999).