From the Western Vascular Society

p27kip1 Knockout enhances collateralization in response to hindlimb

Galit Ankri-Eliahoo, PhD,a Kevin Weitz, BS,a Timothy C. Cox, PhD,b and Gale L. Tang, MD,a,c Seattle, Wash

Objective: The natural response to arterial occlusive disease is enlargement of collaterals; however, the molecular factors that control collateralization are not well understood. The gene p27Kip1 (p27) affects human response to arterial injury. Previous studies have shown that overexpression of p27 inhibits vascular endothelial and vascular smooth muscle cell (VSMC) proliferation and . To test the hypothesis that knockout of p27 would improve collateralization in reaction to L L ischemia, we performed in vivo and in vitro experiments using p27 knockout (p27 / ) and wild-type (wt) mice. L L Methods: Hindlimb ischemia was induced by left femoral artery ligation in p27 / and wt (C57BL/6) female mice. The mice underwent weekly laser Doppler imaging of the footpads until sacrifice on postoperative day 28 followed L L by microcomputed tomography scanning of both hindlimbs. VSMCs were isolated from p27 / and wt mice and used in migration and gel contraction assays in the absence and presence of the nonspecific matrix metalloproteinase (MMP) inhibitor BB94. MMP-2 and MMP-9 messenger RNA (mRNA) expression was measured by quantitative reverse L L transcription-polymerase chain reaction in p27 / and wt VSMCs. L L Results: p27 / mice reperfused more effectively than wt mice by laser Doppler starting from day 7 (ischemic/nonischemic ratio, 0.33 6 0.02 vs 0.25 6 0.02; P < .05) and continuing through day 28 (0.45 6 0.04 vs 0.31 6 0.04; P < .05). The L L gracilis collateral diameter was similar for the nonischemic hindlimbs of the p27 / and wt mice, and this collateral L L pathway increased similarly after ischemia as assessed by microcomputed tomography. However, the p27 / mice significantly enlarged a novel collateral pathway that bridged directly between the femoral artery proximal to the ligation site and the saphenous or popliteal artery distal to the ligation site more than wt mice (158 6 18.3 vs 82 6 22 mm; P < .001). L L p27 / VSMCs migrated more (79% 6 5% vs 56% 6 6%; P < .05) and caused more gel contraction (18% 6 5% of the initial L L area vs 43% 6 4%; P < .05) than wt cells. Migration and collagen contraction were abolished in p27 / and wt cells by MMP L L inhibition. p27 / cells expressed significantly more MMP-2 mRNA than wt cells did. Conclusions: Knockout of p27 enhances arterial collateralization in response to hindlimb ischemia through enlargement of a new collateral pathway. In vitro, knockout of p27 increases collagen gel contraction in addition to stimulating VSMC migration. We speculate that p27 may affect collateralization through its role in regulating MMP-2 expression. (J Vasc Surg 2016;63:1351-9.)

Clinical Relevance: is the leading cause of mortality and morbidity in the United States. _ENREF_2 The adaptive response to the progressive occlusion of arteries is collateralization (). As many patients with severe atherosclerosis are not good candidates for angioplasty or surgical bypass, therapies directed toward improving collat- eralization are needed. The molecular pathways controlling collateralization, however, are not well understood. The human response to arterial injury is affected by a genetic polymorphism in the gene CDKN1B (p27Kip1 or p27). We demonstrate that knockout of p27 improves collateralization. Study of the molecular partners of p27 will identify therapeutic candidates to enhance this process for patients.

Collateralization (arteriogenesis) is the adaptive collaterals results in restoration of approximately 30% of response to the progressive occlusion of arteries caused original flow,1 which is frequently insufficient by atherosclerosis in both the coronaries and the peri- to avoid further interventions to improve distal blood pheral vasculature. At best, however, this enlargement of flow. Thus far, therapies directed toward enhancing

From the Division of Vascular Surgery, University of Washingtona; the Additional material for this article may be found online at www.jvascsurg.org. Department of Pediatrics, University of Washington, and Center for Correspondence: Gale L. Tang, MD, VA PSHCS, Division of Vascular Developmental Biology and Regenerative , Seattle Children’s Surgery, Department of Surgery, University of Washington, Surgical Research Instituteb; and the Division of Vascular Surgery, Department Services 112, 1660 S Columbian Way, Seattle, WA 98108 (e-mail: of Surgery, VA Puget Sound Health Care System.c [email protected]). The content is solely the responsibility of the authors and does not neces- The editors and reviewers of this article have no relevant financial rela- sarily represent the official views of the Department of Veterans Affairs tionships to disclose per the JVS policy that requires reviewers to or the United States Government. decline review of any manuscript for which they may have a conflict Author conflict of interest: none. of interest. Presented as a long presentation at the Twenty-ninth Annual Meeting 0741-5214 of the Western Vascular Society, Coronado Bay, Calif, September Published by Elsevier Inc. on behalf of the Society for Vascular Surgery. 20-23, 2014. http://dx.doi.org/10.1016/j.jvs.2014.12.047

1351 JOURNAL OF VASCULAR SURGERY 1352 Ankri-Eliahoo et al May 2016 collateralization have been disappointing in larger, ran- Femoral A. domized clinical trials,2-4 most likely because the molecular factors that control collateralization are not well Superficial Bridge Collaterals understood.5 Epigastric A. Ligature A genetic polymorphism in the gene for the cell cycle inhibitor CDKN1B, also known as p27Kip1 (p27), affects Artery 1 Gracilis the human response to arterial injury. Patients who are ho- Collateral 1 mozygous for the minor variant A allele of a common Popliteal A. single-nucleotide polymorphism of the promoter region of p27 have decreased coronary in-stent restenosis and Artery 2 60% less chance to experience vein graft failure than pa- Gracilis tients who are heterozygous or homozygous for the C Collateral 2 allele.6,7 The A allele in the promoter was associated with 20-fold increase in reporter gene expression.7 Overexpres- Artery 3 sion of p27 using an adenoviral vector resulted in decreased Gracilis blood flow recovery in a murine hindlimb ischemia model,8 Collateral 3 suggesting a possible role for p27 in collateralization. If it is confirmed, genetic variation in p27 may explain why some patients have abundant collaterals and remain asymptom- atic whereas others manifest severe disease. Fig 1. Schematic diagram showing the arterial anatomy of the To better understand the role of p27 in collateraliza- À/À mouse hindlimb. Indicated on the diagram are the femoral area tion, we tested the collateralization response of p27 position (just proximal to the ligation); artery positions 1 (proximal mice to hindlimb ischemia. We hypothesized that reduced saphenous artery), 2 (midsaphenous artery), and 3 (distal saphe- expression of p27 would improve collateralization in nous artery); gracilis collateral positions 1 (upstream collateral), 2 response to ischemia. We also tested the effect of p27 on (midcollateral), and 3 (downstream collateral); and bridge collat- vascular smooth muscle (VSMC) migration and gel eral positions. contraction in the presence and absence of matrix metallo- proteinase (MMP) inhibition. We further compared messenger RNA (mRNA) expression levels of MMP-2 blanket to 37C under 1.5% isoflurane anesthesia delivered À/À and MMP-9 in p27 and wild-type (wt) cells to provide with 1 L/min O2, were scanned with a MoorLDI2 laser a possible mechanism by which p27 inhibits collateral ar- Doppler perfusion imager (Moor Instruments, Wilming- tery remodeling. ton, Del) immediately postoperatively and on postopera- tive days 7, 14, 21, and 28. Flux values were averaged METHODS for the entire footpad and then expressed as a ratio of ischemic (left)/nonischemic (right). Animal care and procedures Pressure perfusion fixation. All mice subjected to Animal care. All animal experiments were performed hindlimb ischemia underwent laparotomy under 2% isoflur- with approval of the Institutional Animal Care and Use ane anesthesia with 1 L/min O2 on postoperative day 28. Committees of the University of Washington and the VA A 22-gauge silicon intra-aortic catheter was used to deliver Puget Sound Health Care System. p27-deficient female 10 mL of vasodilator solution (1 mM nitroprusside, mice (p27À/À, backcrossed at least nine generations to 10 mM adenosine, 2.5 U/mL heparin in saline) after a nick C57BL/6 background, B6.129S4-Cdkn1btm1Mlf/J, in the inferior vena cava was made to allow outflow.10 weights 22-27 g) and C57BL/6 female mice (wt, weights Pressure perfusion fixation was then carried out at 18-21 g) were obtained from the Jackson Laboratories 100 mm Hg with methyl Carnoy solution. After 2 minutes (Bar Harbor, Me). Female mice were used because they are of fixation, contrast material (60% barium sulfate, liquid E- known to collateralize more poorly than male mice.9 All Z-Paque) was infused through the intra-aortic catheter mice were maintained under standard conditions of 12- until the hindlimb arteries were filled (approximately hour light and dark cycles, with access to chow and water 200 mL per mouse). The hindlimbs then underwent im- ad libitum. mersion fixation with methyl Carnoy overnight, followed Hindlimb ischemia by femoral artery ligation. The by dehydration with 70% EtOH. The hindlimb specimens 3- to 5-month old p27À/À (n ¼ 10) and wt (n ¼ 9) mice then underwent microcomputed tomography (microCT) fl were anesthetized using iso urane with 1 L/min O2. scanning. Buprenorphine (0.05 mg/kg) was given subcutaneously MicroCT scanning and analysis. Hindlimbs (n ¼ 5 for analgesia before ligation. The left femoral artery was p27À/À and n ¼ 4 wt sets of nonischemic and ischemic ligated with 6-0 silk suture just distal to the superficial hindlimbs) were imaged at the Seattle Children’s Research epigastric artery (Fig 1). Institute’s Small Animal Tomographic Imaging Facility. Laser Doppler perfusion imaging. Mouse plantar The imaging was performed with a model 1076 microCT footpads, equilibrated on a homeothermic warming scanner (SkyScan, Kontich, Belgium). Scans were JOURNAL OF VASCULAR SURGERY Volume 63, Number 5 Ankri-Eliahoo et al 1353

Fig 2. p27À/À mice revascularize better than wild-type (wt) control mice. A, Representative serial laser Doppler flux images of the mouse plantar footpads (left, ischemic; right, control) for p27À/À and wt mice measured weekly during 28 days. B, The p27À/À mice (n ¼ 10) had significantly improved blood flow (ischemic/nonischemic ratio) starting at day 7 and continuing through day 28 compared with wt mice (n ¼ 9). *P < .05. POD, Postoperative day. performed at 9-mm resolution using the following settings: vessel number and area from the ischemic leg were normal- 70 kV, 120 mA, 0.5-mm aluminum filter, 550-ms exposure ized to the uninjured leg. with three-frame averaging, 180-degree scan, and a rota- Mouse arterial smooth muscle cell isolation. tion step of 0.7 degree. VSMCs were isolated from mouse aortae as previously pub- Reconstruction of the raw data images was done with lished.11 Briefly, aortae, harvested from 8- to 10-week-old NRecon V1.6.1.0 (SkyScan) software and analyzed with mice (n ¼ 15 wt, three separate isolations, and n ¼ 20 Analyze 10.0 (Mayo Clinic), MATLAB R2013a (Image p27À/À, four separate isolations) were incubated for Processing Toolbox; MathWorks, Natick, Mass), and 10 minutes in an enzyme mix containing 2 mg/mL bovine CTan (SkyScan) as follows. The images were thresholded serum albumin, 0.125 mg/mL elastase type III (Sigma- to a level at which the soft tissue was invisible, and the ves- Aldrich, St. Louis, Mo), 1 mg/mL collagenase CLS, and sels were separated into distinct entities, which limited the 0.375 mg/mL soybean trypsin inhibitor (Worthington, analysis to vessels >25 mm in diameter. The leg bones (fe- Lakewood, NJ) in Hanks balanced salt solution (Gibco Life mur, fibula, tibia, and patella) were digitally removed in Technologies, New York, NY) to remove the adventitial Analyze by the Image Segmentation module. Vessel statis- layer. The remaining tissue was incubated at 37C for a tics were obtained with the MATLAB Image Processing further 2 hours. Cells were collected by centrifugation and Toolbox and CTan’s two-dimensional statistical calcula- placed in media containing 20% fetal bovine serum (FBS) tors. The mean number of vessels and the average size of for expansion. all vessels, in particular the collaterals, popliteal, and saphe- Migration assay. The scratch assay was performed as nous arteries, were compiled from two-dimensional cross described previously.12 In brief, 150,000 cells per well sections of each volume of interest and averaged. The were plated in a 24-well tissue culture plate (Falcon, JOURNAL OF VASCULAR SURGERY 1354 Ankri-Eliahoo et al May 2016

A Non-ischemic Ischemic p27-/-

1 1

L

2

2 B

3 3

wt

1 1

L 2 2

3 3

Fig 3. Bridge collateral diameters significantly increased in p27À/À mice after hindlimb ischemia. A, Representative microcomputed tomography (microCT) scans of the nonischemic and ischemic hindlimbs from a p27À/À and a wild- type (wt) mouse at 28 days after induction of ischemia. The arteries have been digitally pseudocolored to better differentiate the main arteries (1, pink) from defined collateral pathways (2, blue) and other muscle branches (3, red). L, Location of ligation. Bridge collateral pathway denoted with arrow. B, The vessel diameters were measured from the microCT scans (n ¼ 5 p27À/À and n ¼ 4 wt sets of hindlimbs). The graph presents the summary of the different vessel diameters from the ischemic hindlimb. The gracilis collateral diameters were measured at the collateral 1 position, and the bridge collateral diameters were measured at the femoral artery position. *P < .001.

Oxnard, Calif) with Dulbecco modified Eagle medium procedure produced gels approximately 1 mm thick with a (DMEM; Gibco Life Technologies) and 2% FBS and diameter of 15 mm. After polymerization, medium incubated at 37C overnight. A straight wound was created (DMEM þ 10% FBS without or with 10 nM BB94) was by a 1-mL pipet tip, and medium containing DMEM, 5% added to each well. The gels were photographed at t ¼ FBS, 5 mM hydroxyurea to prevent proliferation (Sigma- 0 and t ¼ 20 hours. Photomicrographs were analyzed with Aldrich), and, in some experiments, 10 nM BB9413 the MATLAB image analysis toolbox. (batimastat, a nonspecific MMP inhibitor; British Total RNA extraction and quantitative reverse Biotechnology, Oxford, UK) was added to the wells. A transcription-polymerase chain reaction (qRT-PCR). dose-response curve for BB94 was done (data were not Total RNA was isolated from cell pellets with the RNeasy shown), and cells were viable after treatment. The wound Mini Kit (Qiagen, Valencia, Calif). Complementary DNA area was imaged with a digital camera at t ¼ 0 and t ¼ (cDNA) was synthesized from 5 mg of total RNA with the 20 hours, and the captured photomicrographs were QuantiTect Reverse Transcription Kit (Qiagen). The following measured with the MATLAB image analysis toolbox. primers were used (all 50 to 30 direction): 18S RNA forward Collagen gel contraction assay. Collagen gels were primer GGACCAGAGCGAAAGCATTTGCC and reverse prepared as described previously14 with some modifica- primer TCAATCTCGGGTGGCTGAACGC; p27 forward tions. Bovine type I collagen (BD Biosciences, San Jose, primer GATGGACGCCAGACCAAGC and reverse primer Calif) was adjusted to physiologic ionic strength, pH, and a CTCCTGCCATTCGTATCTTGC; MMP-2 forward concentration of 1 mg/mL with DMEM, NaHCO3 primer ATCTGGTGTCTCCCTTACGG and reverse primer (0.025 M), and NaOH (4.4 mM), while it was maintained TGCAGTGATGTCCGACAAC; and MMP-9 forward at 4C. The wells of a 24-well tissue culture plate (Falcon) primer GCATACTTGTACCGCTATGG and reverse primer were coated with a thin layer of 1% agarose (Sigma- TGTGATGTTATGATGGTCCC. All primers were pur- Aldrich) solution. Collagen mix solution, 1 mL, was added chased from Santa Cruz Biotechnology (Santa Cruz, Calif). to 3 Â 105 cells and then pipetted into the precoated wells The qRT-PCR of MMP-2, MMP-9, and p27 mRNA in and allowed to polymerize at 37C for 90 minutes. This total RNA from wt cells and p27À/À cells was performed JOURNAL OF VASCULAR SURGERY Volume 63, Number 5 Ankri-Eliahoo et al 1355 with RT2 SYBR Green ROX qPCR Mastermix (Qiagen). A PCR reactions were set up in a volume of 10 mL, in which 1 mL of synthesized cDNA was included. Each reaction con- tained SYBR Green master mix, 1 mM of forward primer, and 1 mM of reverse primer. DNA amplifications were car- ried out in a 96-well reaction plate format with a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, Calif). Each sample was analyzed in triplicate. Mouse 18 S ribosomal RNA was evaluated in all samples as an inter- nal control. Thermal cycling was initiated with a 2-minute incubation step at 50C, followed by an initial denaturation step of 10 minutes at 95C, and then 40 cycles of 95C for number in vessel % Increase 15 seconds and 60C for 1 minute were carried out. A disso- ciation stage was added: 95C for 15 seconds followed with   60 C and 95 C for 15 seconds each. The Ct (cycle threshold) was defined as the number of cycles required for the fluores- B cent signal to cross the threshold. The relative amount of gene mRNA expression to 18S was calculated by the for- mula DDCt ¼ Ctwt (gene) À Ctwt (18S) À Ctko (gene) À Ct (18S). The relative amount of mRNA expression was ko DD expressed as a percentage of wt expression, % ¼ 100 * 2 Ct. Transfection of small interfering RNA (siRNA) targeting p27. The wt VSMCs were transfected with 5 nM siRNA targeting p27 or scrambled siRNA (Santa Cruz Biotechnology) using the Amaxa Human AoSMC Nucleofector Kit (Lonza, Portsmouth, NH) according to area in vessel % Increase the manufacturer’s instructions. Cells were suspended in 10% FBS in DMEM, left overnight, and changed to 10% FBS in DMEM with penicillin-streptomycin for 24 hours before use. Percentage knockdown was determined by qRT-PCR analysis for p27 from mRNA isolated from both C transfection groups. Statistical analysis Results are expressed as mean 6 standard error of the mean. Differences between two groups were analyzed by the Student t-test. P < .05 was used to determine statistical significance. Laser Doppler imaging analysis results were tested by within-subject modeling and unpaired t-test. Number of vessels Fisher exact test was used to determine significance of detection vs nondetection of bridge collaterals in the non- ischemic hindlimbs.

Fig 4. After hindlimb ischemia, p27À/À mice have increased RESULTS arterial density. A, Arterial number, as measured by micro- L L Improved blood flow recovery in p27 / mice af- computed tomography (microCT) at the femoral artery position ter femoral artery ligation. Fig 2 shows the results of laser of the ischemic hindlimb and expressed as percentage increase À/À Doppler perfusion imaging of p27À/À and wt mice during over the nonischemic hindlimb, increased more in the p27 ¼ ¼ < 28 days. Fig 2, A shows representative perfusion images for mice (n 5) than in the wild-type (wt)mice(n 4). *P .03. À À À À B, Graph showing the total arterial area as a percentage of total a p27 / and a wt mouse. The p27 / mice had signifi- fl cross-sectional area (both normalized to the uninjured hin- cantly more blood ow recovery than wt mice starting at dlimb) at the femoral artery position. *P < .05. C, Graph postoperative day 7 and continuing through day 28 (Fig 2, quantitating the number of arteries at the femoral position of B). No digital or foot necrosis was noted in either strain. the ischemic hindlimb above and below 70 mm. The p27À/À There were no significant differences between the two mice had significantly more arteries larger than 70 mmthanthe groups for the nonischemic hindlimb at any time point. wt mice did. *P < .02. JOURNAL OF VASCULAR SURGERY 1356 Ankri-Eliahoo et al May 2016

A p27-/- wt

t=0

B -BB94

t=20 hrs

-/- +BB94

t=20 hrs

siRNA p27 knock-down siRNA scrambled

-BB94

t=20 hrs

Fig 5. Decreasing p27 expression in vascular smooth muscle cells (VSMCs) increases migration. A, Under conditions of growth arrest, the top four panels show representative photomicrographs of a scratch assay using confluent VSMCs derived from p27À/À (left panels) and wild-type (wt) aortae (right panels) in the absence and presence of BB94. The bottom panels show a scratch assay using wt VSMCs transfected with small interfering RNA (siRNA) directed against p27 (left panel) or scrambled siRNA (right panel). The dotted lines mark the edge of cell migration. B, Graph demonstrating the increased migration by p27À/À cells. The wt cells expressing less p27 (through siRNA knockdown) also migrate more than wt cells transfected with scrambled siRNA. BB94 inhibits cell migration by both p27À/À and wt cells. Values are expressed as the percentage of the scratch area after 20 hours from the initial scratch area at t ¼ 0. n ¼ 4 separate experiments using two different cell lines for each group. *P < .01.

Increased bridge collateral diameters after hindlimb it originates from the femoral artery immediately proximal L L ischemia in p27 / mice. Representative images from to the ligation site (schematically diagrammed in Fig 1) microCT scans at postoperative day 28 for a p27À/À and is significantly shorter than the gracilis collateral and a wt mouse are shown in Fig 3, A. Movies of the pathway (3.7 6 1.1 mm vs 8.6 6 .5 mm; P < .001). three-dimensional reconstructions are available online The diameter of the baseline p27À/À bridge collaterals, (Supplementary Video, online only). The popliteal and as detected in the nonischemic hindlimb, trended to be saphenous artery diameters for the nonischemic leg were slightly larger than the wt baseline bridge collaterals not significantly different between the p27À/À and wt mice (67 6 14 p27À/À vs 54 6 10 wt; P ¼ .08). In the (243 6 30 vs 239 6 31 mm; P ¼ .43), despite the overall ischemic hindlimb, the bridge collaterals of p27À/À increased size of the p27À/À mice (average weight, 23.9 6 mice enlarged significantly more than those of the wt 1.5 vs 19.7 6 0.9 g; P < .0001). Likewise, there was no mice (158 6 18 vs 82 6 22 mm; P < .001). At baseline, difference in the diameters of the well-described gracilis the p27À/À mice may have more robust collaterals than and superficial dorsal collateral pathways after femoral ar- the wt mice, as we detected four major collaterals in the tery ligation.10 The diameter of the gracilis collaterals was nonischemic hindlimb in all five of the p27À/À mice and similar for the nonischemic hindlimb of the p27À/À and wt only two of the four wt mice, although this difference mice (75 6 19 vs 60 6 16 mm; P ¼ .095, respectively), and was not statistically significant (P ¼ .17 by Fisher exact it enlarged in both equivalently after ischemia (98 6 18 vs test). This possible baseline difference did not affect func- 106 6 17 mm; P ¼ .31; Fig 3, B). tional blood delivery as there was no difference by laser The p27À/À mice, however, significantly increased the Doppler perfusion imaging between the two groups size of a collateral pathway that has not previously been immediately after ligation (ischemic/nonischemic ratio, described. We named this pathway “bridge collateral” as 0.078 6 0.004 p27À/À vs 0.054 6 0.004 wt; P ¼ .45). JOURNAL OF VASCULAR SURGERY Volume 63, Number 5 Ankri-Eliahoo et al 1357

A

B

Fig 6. p27À/À vascular smooth muscle cells (VSMCs) cause more collagen gel contraction than wild-type (wt) VSMCs do. A, Representative photomicrographs from a collagen gel contraction assay. p27À/À VSMCs were suspended in a collagen gel in the absence and presence of BB94. The pink dashed circles represent the circumference of the collagen gel area measured at time 0 and 20 hours. B, Graph demonstrating the increased collagen gel contraction caused by p27À/À cells, which is inhibited by BB94. Values are expressed as the percentage of collagen gel area after 20 hours from the initial collagen gel area at t ¼ 0. n ¼ 4 experiments. *P < .05.

L L Arterial density after hindlimb ischemia increased Increased migration and gel contraction by p27 / L L in p27 / mice. To determine whether there was an VSMCs inhibited by MMP inhibitor. To confirm that increase in arterial density, we counted the number of p27 affects VSMC motility,15 we examined the ability of arteries at defined locations along the arterial tree p27À/À and wt VSMCs to migrate in a standard cell culture (femoral, artery position 1, artery position 2, and artery migration assay. After 20 hours, more p27À/À VSMCs than position 3 as indicated on Fig 1) and also measured the wt VSMCs migrated into the denuded region (Fig 5)as ratio of the arterial wall area relative to the whole cross- measured by a decrease in wound area. The wt VSMCs sectional area. We normalized both measurements to treated with siRNA directed against p27 (knockdown effi- the nonischemic hindlimb to control for the difference ciency 70%) likewise migrated more effectively than wt in size between the two strains. The nonischemic hin- VSMCs treated with scrambled siRNA. The assay was done dlimbs had a similar number of arteries identified for in the presence of hydroxyurea for growth arrest, so the both groups (p27À/À 66 6 16 vs wt 55 6 10; P ¼ .12) decrease in wound area was solely the result of cell migra- and a similar vessel to total area (p27À/À 1.4% 6 0.3% tion and not of cell proliferation. We repeated the migration and wt 1.2% 6 0.2%; P ¼ .1). There were significant experiment in the presence of the nonspecific MMP in- differences between the two groups after ischemia only hibitor BB94 (Fig 5, A and B) and found that cell migration at the femoral artery position. Fig 4, A shows that the was completely abolished for both genotypes with MMP ratio of vessels between the ischemic/nonischemic inhibition. hindlimb increased significantly more for the p27À/À We also measured the influence of p27 on collagen gel than for the wt mice. This difference was also significant contraction. Fig 6, A shows photomicrographs of represen- when measured as vessel area at the femoral level relative to tative collagen gels (light pink circles) containing either total tissue area (Fig 4, B). Fig 4, C shows that the increase p27À/À or wt VSMCs. The p27À/À VSMCs caused more in number of arteries in the p27À/À mice was due to gel contraction than wt VSMCs did (Fig 6, B). Again, development of larger arteries as the p27À/À mice had this effect was abrogated by the addition of BB94 (Fig 6, significantly more arteries with a diameter >70 mm. A and B). L L There was no significant difference between the p27À/À MMP-2 mRNA expression increased in p27 / and wt mice in terms of number of arteries with a VSMCs. To better define the effect of p27 knockout on diameter <70 mm(P > .2). MMP expression, we examined mRNA expression by JOURNAL OF VASCULAR SURGERY 1358 Ankri-Eliahoo et al May 2016

the cytoplasm and that this response might be dependent on regulation of MMPs. However, we cannot eliminate the possibility that increased proliferation is also neces- sary for improved collateralization. Distasi et al10 previously described deep adductor, mid- dle dorsal, and superficial dorsal collateral pathways induced in response to hindlimb ischemia by using Microfil casting. Using microCT, we detected a new, more efficient collateral pathway in the p27À/À mice. The bridge collat- erals were the dominant collateral pathway in the p27À/À mice and enlarged significantly more than in wt mice, whereas the gracilis collateral pathway increased equally in both the p27À/À and wt mice. We therefore concluded that these bridge collaterals had significant influence on the improved blood flow in the p27À/À ischemic hindlimbs. À/À Fig 7. p27 vascular smooth muscle cells (VSMCs) express The finding that MMP inhibition prevents the fi signi cantly more matrix metalloproteinase 2 (MMP-2) messenger enhanced migration seen in p27À/À cells is new, as is the RNA (mRNA) than wild-type (wt) cells do. The left bars À À finding that p27 / cells overexpress MMP-2. It has demonstrate that the p27 mRNA expression is effectively knocked out in the isolated p27À/À VSMCs. The right bars show that the been previously shown that migration of VSMCs derived p27À/À VSMCs express significantly more MMP-2 mRNA than from aortae of various species and human saphenous vein wt cells do. Relative p27 mRNA expression was normalized to is inhibited by synthetic MMP inhibitors and by gene trans- 100% for the wt cells for both genes. *P < .001. fer of tissue inhibitors of matrix metalloproteinase (TIMP- 1, TIMP-2, and TIMP-3).22,23 MMP-9 has been shown both to increase VSMC-induced collagen contraction at qRT-PCR of the two MMPs, MMP-2 and MMP-9, pre- low concentrations and to inhibit it at higher concentra- viously demonstrated to affect arterial remodeling16 in tions.24 Stretch and mechanical injury upregulate MMP-2 p27À/À and wt VSMCs. We also confirmed that the p27À/ and MMP-9 production.25,26 In contrast to our findings, À VSMCs express very little p27 mRNA (Fig 7). MMP-2 another group found that MMP inhibition did not prevent mRNA expression was significantly increased in p27À/À collagen gel contraction by cynomolgus monkey aortic cells compared with wt cells (Fig 7). The mRNA expression cells.27 However, the molecular mechanism by which of MMP-9, however, was similar in the p27À/À and wt p27 regulates MMPs and through which MMP affects cells (data were not shown). This was not unexpected as migration, proliferation, and collagen gel contraction in MMP-9 is generally upregulated only after arterial injury. VSMCs is not completely clear. There are several limitations to this study. First, we have DISCUSSION not confirmed that p27À/À mice at baseline do not have a We demonstrated that p27À/À mice revascularize more different pattern of collaterals than wt mice, although we effectively than wt mice in response to hindlimb ischemia. did not detect any differences between the two strains in This was based on both increased functional downstream the nonischemic leg. Second, we only examined collaterali- perfusion as measured by laser Doppler perfusion imaging zation at a relatively late time point and did not examine and increased enlargement of a more efficient (shorter capillary angiogenesis, which previous work suggests is and more direct) bridge collateral pathway as measured affected by p27 expression.8 Therefore, we cannot definitely by microCT in the p27À/À mice. We further showed that conclude that the improvement in blood flow recovery in decreasing expression of p27 in VSMCs allows them to p27À/À mice is only due to improved bridge collateraliza- migrate more efficiently and to cause more collagen gel tion. Third, because the limit of resolution of our microCT contraction. Both migration and collagen gel contraction technique was 25 mm, we cannot determine whether new are abolished by nonspecific MMP inhibition. The differ- vessels formed in response to hindlimb ischemia or if they ences in migration and collagen gel contraction seen with just enlarged above the threshold of detection. As the major- decreased p27 expression may be secondary to increased ity of collaterals were also detected in the nonischemic limb, MMP-2 expression. the second possibility seems more likely. Fourth, BB94 is a Our current knowledge about the molecular path- broad inhibitor of zinc-dependent MMPs,28 and so it may ways through which p27 affects the arteries is poor. It have had additional inhibitory effects than just on MMPs. is known that p27 functions in the nucleus to regulate Last, although p27 has been shown to have effects on cell proliferation and in the cytoplasm for motility and bone marrow progenitors and leukocytes and on cell prolif- cell contact with the extracellular matrix.17-20 It has eration, we did not examine early time points to determine been previously shown that p27 inhibits cell proliferation whether there was increased arterial wall cell proliferation and that p27À/À VSMCsproliferatebetterthanwt or increased monocyte infiltration or proliferation in the VSMCs.21 OurresultsinVSMCssuggest that the effect p27À/À mice. We plan to pursue these experiments in the of p27 on collateralization may be through its role in future to determine whether p27’s effect on collateralization JOURNAL OF VASCULAR SURGERY Volume 63, Number 5 Ankri-Eliahoo et al 1359

is due to decreased MMP-2 expression, effects on bone 12. Mason DP, Kenagy RD, Hasenstab D, Bowen-Pope DF, Seifert RA, marrow-derived cells, or arterial wall cell proliferation. Coats S, et al. Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the CONCLUSIONS injured rat carotid artery. Circ Res 1999;85:1179-85. 13. Zempo N, Koyama N, Kenagy RD, Lea HJ, Clowes AW. Regulation of Knockout of p27 enhances arterial collateralization in vascular smooth muscle cell migration and proliferation in vitro and in response to hindlimb ischemia through enlargement of a injured rat arteries by a synthetic matrix metalloproteinase inhibitor. new, more efficient bridge collateral pathway. This enlarge- Arterioscler Thromb Vasc Biol 1996;16:28-33. 14. Li S, Moon JJ, Miao H, Jin G, Chen BP, Yuan S, et al. Signal ment may be secondary to increased MMP-2 activity in the transduction in matrix contraction and the migration of vascular À/À p27 VSMCs. smooth muscle cells in three-dimensional matrix. J Vasc Res 2003;40: 378-88. This work was supported with the facilities and re- 15. Diez-Juan A, Castro C, Edo MD, Andres V. Role of the growth sup- sources of the VA Puget Sound Health Care System, the pressor p27Kip1 during vascular remodeling. Curr Vasc Pharmacol Seattle Institute for Biomedical and Clinical Research, the 2003;1:99-106. University of Washington Royalty Research Fund, the 16. Whatling C, McPheat W, Hurt-Camejo E. Matrix management: Vascular Cures Foundation, and the Laurel Foundation. assigning different roles for MMP-2 and MMP-9 in vascular remod- eling. Arterioscler Thromb Vasc Biol 2004;24:10-1. 17. Baldassarre G, Belletti B, Nicoloso MS, Schiappacassi M, Vecchione A, AUTHOR CONTRIBUTIONS Spessotto P, et al. p27Kip1-stathmin interaction influences sarcoma cell migration and invasion. Cancer Cell 2005;7:51-63. Conception and design: GE, KW, TC, GT 18. McAllister SS, Becker-Hapak M, Pintucci G, Pagano M, Dowdy SF. Analysis and interpretation: GE, KW, TC, GT Novel p27kip1 C-terminal scatter domain mediates Rac-dependent cell Data collection: GE, KW, GT migration independent of cell cycle arrest functions. Mol Cell Biol Writing the article: GE, GT 2003;23:216-28. Critical revision of the article: GE, KW, TC, GT 19. Soos TJ, Kiyokawa H, Yan JS, Rubin MS, Giordano A, DeBlasio A, et al. Formation of p27-CDK complexes during the human mitotic cell Final approval of the article: GE, KW, TC, GT cycle. Cell Growth Differ 1996;7:135-46. Statistical analysis: GE, GT 20. Vidal A, Koff A. Cell-cycle inhibitors: three families united by a com- Obtained funding: GT mon cause. Gene 2000;247:1-15. 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