ZFP148 binds cooperatively with NF-1 to inhibit Smooth Muscle Marker Gene Expression during Abdominal Aortic Aneurysm Formation

Abstract

Objective:

The goal of this study was to determine the role of ZFP148 in aneurysm formation.

Approach and Results:

ZFP148 mRNA expression increased at day 3, 7, 14, 21 and 28 following during AAA formation in C57BL/6 mice. Loss of ZFP148 conferred AAA protection using ERTCre+ ZFP148 flx/flx mice. In a third set of experiments, smooth muscle specific loss of ZFP148 alleles resulted in progressively greater protection using novel transgenic mice (MYHCre+ flx/flx, flx/wt, and wt/wt). Elastin degradation, LGAL3, and Neutrophil staining were significantly attenuated, while α-actin staining was increased in ZFP148 knock-out mice. Results were verified in total cell ZFP148 and smooth muscle specific knock-out mice using an Angiotensin II model, .ZFP148 smooth muscle specific conditional mice demonstrated increased proliferation and ZFP148 was shown to bind to the p21 promoter during AAA formation. ZFP148 smooth muscle specific conditional knock-out mice also demonstrated decreased apoptosis as measured by decreased cleaved Caspase-3 staining. ZFP148 bound smooth muscle marker genes via ChIP analysis mediated by NF-1 promote histone H3K4 de-acetylation via Histone De-acetylase 5. Transient transfections and ChIP analyses demonstrated that NF-1 was required for ZFP148 protein binding to Smooth Muscle marker genes promoters during aneurysm formation. Elimination of NF-1 using shRNA approaches demonstrated that NF-1 is required for binding and elimination of NF-1 increased BRG1 recruitment, the ATPase subunit of the SWI/SWF complex, and increased histone acetylation.

Conclusions:

ZFP148 plays a critical role in multiple murine models of aneurysm formation. These results suggest that ZFP148 is important in the regulation of proliferation, smooth muscle gene down-regulation and apoptosis in aneurysm development.

Introduction

Abdominal aortic aneurysms (AAA) are a major health threat in the United States as they remain the 15^th leading cause of death, specifically affecting men 4:1 over women in the currently aging population^1–4. Even more concerning, AAAs results in more than 15,000 surgical procedures annually as there is currently no medical treatment strategy for slowing the growth rates of this deadly disease^1–4. Aortic aneurysms have been characterized initially by elevation of pro-inflammatory cytokines associated with elastin and collagen degradation. In addition, Smooth muscle cell apoptosis coupled with collagen and elastin degradation lead to the continued pathogenesis of the disease^5–7. While many studies have examined the mechanisms of inflammatory cell infiltration during AAA formation, few studies have examined the detailed mechanism by which vascular smooth muscle cells (VSMCs) are impacted during AAA formation⁸. The effects of VSMC migration and apoptosis accompanied by extracellular matrix (ECM) degradation is believed to be the major processes leading to the weakening of the aortic wall and subsequent aneurysm formation^{9, 10}. However, the mechanisms that regulate this complex process and the cell-cell interactions remain unknown. To date, there are no medical therapies available to slow its progression once the disease is diagnosed.

Smooth muscle cells comprise part of the aortic wall and are specifically designed to play a central role in organ function under physiological and pathophysiological conditions. SMCs have been found to be remarkably plastic and are able to respond to a variety of external and internal stimuli including PDGF-BB, DD, POVPC, RA and IL-1^11–15. This process is known as “phenotypic switching” and was defined as the process whereby SMCs transition from their usual, quiescent contractile state to a semi-proliferative-migratory state^{16, 17}. Key to this process is the coordinated down-regulation of markers of differentiated VSMCs, including transgelin, myosin heavy chain and α-actin, genes required for VSMC contraction¹⁶. The down-regulation of these genes is believed to be a key indicator of the process of “phenotypic switching” and, furthermore, the regulatory factors that affect this process are believed to be key regulatory mechanisms of vascular disease¹⁶. It has recently been demonstrated that VSMC “phenotypic switching” plays a key role in the development and progression of atherosclerotic lesions, and regulation of plaque stability by regulating cap formation^{18, 19}. Our laboratory was the first to demonstrate that VSMC “phenotypic switching” was an early event in AAA formation and that the Kruppel-like zinc finger transcription factor (KLF4) plays a role in VSMC phenotypic modulation^{8, 20}. However, there is currently little information, besides our own studies, to describe the mechanism of smooth muscle cell phenotypic switching in the context of AAA formation⁸.

ZFP148 is a Kruppel-type, zinc-finger transcription factor that is closely related structurally to the Specificity protein (Sp) family of transcription factors. ZFP148 has been demonstrated to bind to the same or similar promoter binding sites as the Sp transcription factor family and can act as a competitor for GC-rich promoter elements^21–29. Previously ZFP148 was shown to be a key factor required for apoptosis and macrophage polarization during atherosclerotic lesion development by binding and repressing the Trp53 gene promoter³⁰. However, no studies have investigated the role of ZFP148 in VSMCs during aneurysm disease. Herein, we document that ZFP148 expression is induced in two different models of AAA in mice, and that conditional global- or VSMC-specific knockout of this gene markedly attenuated AAA formation. Identifying ZFP148 as a potential, novel therapeutic target in AAA is a key finding of this report.

Materials and Methods

The authors declare that all supporting data are available within the article and its online supplementary files but can provide additional information upon request to corresponding author.

Defining sex differences as an important biological variable in AAA models:

Male and female mice possess differential abilities to form AAAs based on gender and a number of current unknown variables. C57/B6 are regularly used because porcine pancreatic elastase perfusion induces robust AAAs with diameters approximately 100% larger than a control proximal segment. In contrast, female C57/B6 do not produce AAAs, i.e. the diameter is not 50% larger than a control proximal segment. Therefore, we focused our study on male mice to be able to assess whether deletion of ZFP148 attenuates AAA formation.

Defining ZFP148 in the murine elastase aneurysm model:

Two groups of 8–12 week old wild-type (WT) C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) underwent aortic perfusion with either porcine pancreatic elastase(1 unit/mL) or saline perfusion (WT elastase, n=12/group; WT saline n=12/ group) as described and performed by Johnston et al., and harvested at days 0, 3, 7, 14, 21, and 28 following surgery^{31, 32} Infrarenal abdominal aortas from both groups were evaluated and harvested for tissue analysis by qPCR(n=6/group) and immune-histochemistry(n=6/group). * indicates whether elastase perfusion was significant with a p-value less than 0.05 over saline perfusion by student’s t-test at each individual timepoint.

Creation of ZFP148 transgenic mice

ZFP148 flx/flx ERT Cre- mice and ZFP148 flx/flx ERT Cre+ mice were created by breeding ERT² tamoxifen Cre mice with ZFP1484 flx/flx mice²⁴. These mice are knock-outs of ZFP148 in all cell types and are necessary to use as global knock-out mice are embryonic lethal due to defective spermatogenesis in males³³. When performing experiments, ZFP148 flx/flx ERTCre+ mice and their sibling age matched controls were used at group numbers indicated.. To induce recombination, these mice underwent a series of 10 tamoxifen injections at 6–8 weeks followed by 14 days of rest to wash out any residual tamoxifen. Tamoxifen has been shown to inhibit AAA formation, thus the 14 day wash-out period allows for sufficient tamoxifen removal from the murine system. These mice then underwent porcine pancreatic elastase perfusion(1 unit/mL) followed by harvest 14 days later.

ZFP148 flx/flx MYHCre+, flx/wt MYHCre+, and wt/wt MYHCre+ mice were created by breeding MYH11 Cre+ mice⁵ with ZFP148 flx/flx mice³⁴. These mice were then back-bred together to produce the experimental and control ZFP148 MYHCre+ mice. The MYHCre+ transgene has been used successfully in the past to demonstrate specific smooth muscle specific knock-out of various genes following tamoxifen injections^{19, 35}. The MYH11 Cre transgene is also located on the Y-chromosome, therefore, only male mice can have the Cre gene and be used in the AAA studies. The mice underwent 10 tamoxifen injections, wash-out, and porcine pancreatic elastase perfusion as mentioned for the ERTCre+ mice above.

ZFP148 flx/flx, flx/wt and wt/wt MYHCre+ ApoE−/− mice were created by breeding MYHCre+ ZFP148 flx/flx mice onto an ApoE−/− background. These mice were then back-bred together to create ZFP148 MYHCre+ ApoE−/− mice which were then bred together to produce the experimental MYHCre+ ZFP148 flx/flx and flx/wt ApoE−/− mice and their WT controls. These mice also underwent the same series 10 tamoxifen injections, two weeks of rest and Angiotensin II infused osmotic pump insertion as mentioned in detail below²⁰.

Murine Elastase Model

10 to 12-week male mice were injected with intraperitoneal (IP) ketamine solution, perfused with either porcine pancreatic elastase(1 unit/mL) or saline, allowed to recover, and harvested by Johnston et al.^{31, 32}. The aortas (or aneurysms, when present) were harvested, and either: 1) snap frozen in liquid nitrogen for analyses by real-time polymerase chain reaction (qPCR) or protein extraction, or 2) incubated overnight for histology or immunohistochemistry. Animal care and use were in accordance with the Guide for the Care and Use of Laboratory Animals. The animal protocol was approved by the University of Virginia Institutional Animal Care and Use Committee (#3634) in compliance with the Office of Laboratory Animal Welfare.

Statistical analysis of aortic diameters was performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Maximal aortic dilation (%) was calculated as [maximal aortic diameter – internal control diameter] ÷ internal control diameter * 100%. The internal control was a small segment of normal proximal abdominal aorta just distal to the renal arteries that was above the proximal extent of the AAA. This section was not perfused with elastase, but it was susceptible to blood pressure changes from volume loss during the harvest as well as expected growth with the animal over time. Values are reported as mean ± standard error of the mean. Aortic dilation between groups was compared using a student’s t-test for 2 sample groups or ANOVA for 3 or more sample groups. Post hoc Tukey correction was applied to determine the significance of individual comparisons with α=0.05².

Angiotensin II Infusion

Osmotic pumps (Alzet^® 2004, Durect Corp., Cupertino, California) containing Ang II (1000 ng/kg/min, Sigma Aldrich Inc., St. Louis, Missouri) were introduced into 10 to 12 -week-old ZFP148 flx/flx, flx/wt and wt/wt ApoE−/− male mice as previously described^{31, 36–39}. Mice were housed and maintained at 70°F, 50% humidity, in 12-hour light-dark cycles per institutional animal protocols. All mice were fed ad libitum water and placed on high fat diet (TD 88137, Harlan Teklad Inc., Indianapolis, Indiana) with no restrictions on movement. Aneurysmal segments of the aortas (proximal to the renal arteries) were harvested after 28 days, aneurysm absence or presence was determined and aneurysm type was assessed^{40, 41} and processed for histology or cytokine array analysis. At day 28, video micrometry measurements of the aortic wall diameter were performed in situ using a Q-Color3 Optical Camera (Olympus Corp., Center Valley, Pennsylvania) using QCapture Pro Software version 6.0 (QImaging Inc., Surrey, Canada). Kaplan-meier curves and log-rank (Mantel-Cox) tests tracked the percentage survival of mice over the 28 day period.

In a second model of Angiotensin II infusion, osmotic pumps containing Angiontensin II were introduced into 10 to 12-week-old ZFP148 flx/flx ERTCre+ and ZFP148 flx/flx ERTCre- ZFP148 flx/wt male mice as previously described⁴². Following Angiotensin II infusion, mice received biweekly injections of a TGFβ neutralizing antibody(R and D systems)⁴². These injections allow for aneurysms to form in C57/B6 mice without back-breeding onto and ApoE−/− or LDLR−/− background. After 28 days, aneurysmal segments were harvested as mentioned in the previous paragraph and percentage survival was tracked and log-rank (Mantel-Cox) tests were performed.

mRNA isolation:

mRNA and protein from WT samples(n=6/group) was extracted from frozen aortic tissue samples with TRIzol reagent (Invitrogen, Life Technologies, Grand Island, NY) as described per the manufacturer’s protocol^{20, 31}

Human Tissue Harvest:

Normal abdominal aortas were obtained from transplant donors(n=10/group) and aneurysmal aortas(n=10 male/group; n=6 female/group) were taken from patients undergoing elective open abdominal aortic aneurysm repair. Aortas from patients with known collagen vascular disease were excluded. Aortic tissue specimens were explanted, placed on ice, and then immediately snap-frozen in liquid nitrogen or placed in 10% formalin for histological processing. Collection of human tissue was approved by the patient’s written consent in compliance with the University of Virginia’s Human Subjects Review Committee (HSR #13178).

Real time reverse transcription PCR analysis:

Using isolated mRNA from the mouse aortas and aortic smooth muscle cells, cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time reverse transcription PCR (RT-PCR) was performed using Sensifast SYBR Supermix (Bioline, Taunton, MA) with primers described previously^{20, 43–46}. Target DNA was analyzed with Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA) to obtain melt curves and takeoff values. Levels of mRNA were standardized either with U6 or 18s mRNA, which served as a housekeeping genes for comparison. All experiments were run three times in triplicate unless otherwise mentioned.

Histology:

Murine aortas were harvested at sacrifice for histology analysis after undergoing left ventricular puncture and 4% paraformaldehyde (PFA) antegrade perfusion at physiologic pressure. Further fixation was achieved by overnight incubation in 4% PFA at 4°C followed by paraffin embedding and sectioning at 5μm. After microwave antigen retrieval, antibodies were bound and detected using VectaStain Elite Kit (Vector Laboratories Inc., Burlingame, California). Antibodies for IHC staining were anti-rat Mac2/LGAL3 for macrophages (1:10,000; Cedarlane Laboratories, Burlington, Canada), anti-mouse anti-Neutrophil (Ly 6B.2) for neutrophils (1:10,000; AbD Serotec, Oxford, United Kingdom), anti-goat MMP2 for matrix metalloproteinase-2 (1:350; R&D Systems, Minneapolis, Minnesota), anti-goat MMP9 for matrix metalloproteinase-9 (1:400; R&D Systems, Minneapolis, Minnesota), anti-cleaved caspase 3 for apoptosis (1:100 Cell Signaling Technology), Ki-67 for proliferation(1:100; Abcam, Cambridge, MA), CD45 for B-cells (1:100; eBiosciences), Mast cell protease for Mast cells(1:100, Santa Cruz Biotechnology Inc., Santa Cruz, California) and anti-mouse SMαA for smooth muscle α-actin (1:1000; Abcam, Cambridge, MA). Visualization color development was completed using diaminobenzidine (Dako Corporation, Carpinteria, California) for SMαA, Mac2, anti-neutrophil, CD3ε, MMP2, CD45, KI-67, Mast cell protease, Cleaved caspase 3 and MMP9.

Human confocal immunoflourescent staining was performed for ZFP148 (1:1000, Abcam, Cambridge, Massachusetts USA) dilution followed by TSA amplification using a TSA kit (Invitrogen), SMA for smooth muscle cells (1:1000, Cy3, Sigma Alrich Corporation), S100A4 for adventitial fibroblasts (1:100 Cell Signaling Technology), CD68 for macrophages (Santa Cruz Biotechnology Inc., Santa Cruz, California) and DAPI (1:10,000; Invitrogen).

For mouse confocal staining, ZFP148 (1:1000, Abcam, Cambridge, Massachusetts USA), p21 (1:100, Abcam, Cambridge, MA), BAK (1:100, Santa Cruz Biotechnology Inc., Santa Cruz, California), CD45 for B-cells (1:100; eBiosciences), CD3 for T-cells (1:100, Santa Cruz Biotechnology), LGAL3 for macrophages and nuclei using DAPI (1:10,000; Invitrogen).

Images were acquired using AxioCam Software version 4.6 via 10X, 40X, and 100X objectives and an AxioCam MRc camera (Carl Zeiss Inc., Thornwood, New York). Threshold gated positive signal was detected within the AOI and quantified using Image-Pro Plus version 7.0 (Media Cybernetics Inc., Bethesda, Maryland). Elastin depletion was quantified by counting the number of breaks per vessel and then averaged and graphed. Images were quantified and counted using two independent observers and are graphed as the mean +/− standard deviation.

WST1 Proliferation Assays

The metabolic activity was monitored by the colorimetric WST-1 assay of the mitochondrial dehydrogenases according to the manufacturer’s instructions (11644807001; Roche Applied Science, Rotkreuz, Switzerland). Primary mouse abdominal aortic smooth muscle cells were plated in 96 well plates at 60% confluency and incubated overnight at 37°C. Smooth Muscle cells were then washed in 1X PBS and media switched to serum free media containing DMEM-Ham’s F-12 medium supplemented with 5 μg/ml transferrin, 6.25 ng/ml sodium selenite, 1 μm/l insulin, and 0.2 mmol/l l-ascorbic acid. 24 hours later, cells were treated with either Ad-Empty, Ad-ZBP89, siControl or siZFP148. 24 hours later cells were incubated with either porcine pancreatic elastase (1 u/mL, E7885; Sigma Aldrich, Darmstadt, Germany) or PDGF-BB (10 ng/mL, R and D Systems, Minneapolis, Minnesota USA) and allowed to incubate overnight at 37°C. The following day, media was removed, cells were washed in 1X PBS and then 10 μl of WST-1 reagent was added to 100 μl of serum-free media and allowed to incubate for 4 h. The WST-1 cleavage product was measured for both cells after 2 h at 450 nm (sample) and at 650 nm (background). WST-1 plus medium alone served as a blank, which was subtracted from all values⁴⁷. Percentages of WST-1 activity were calculated by the following formula: (WST-1 value/control) × 100. Results are the average of three separate experiments performed in triplicate.

Mouse aortic smooth muscle cell cultures:

Mouse abdominal aortic smooth muscle were isolated and cultured as previously described^{2, 16}. Western blot analysis was performed at passage 6 to verify that SM-actin, SM22, and SM-MHC were expressed. For siRNA transfections, cells at passages 8–10 were plated in all 6 wells of a 6 well plate at 1×10⁵ cells per well; 24 hours later cells were transfected with either a single siRNA to mouse ZFP148 or a non-targeting control. 24 hours later mouse aortic smooth muscle cells were stimulated by porcine pancreatic elastase(1 unit/mL; Sigma Aldrich), PDGF-BB(R and D Systems; Minneapolis, Minnesota, USA) or representative vehicle for 24 hours². Cells were harvested and RNA was extracted using the TriZol method and qPCR was performed as described above^{44–46, 48}.

Migration assays.

Cell migration assays were performed on Millipore MultiScreen-MIC plates containing 8 μm pores. Cells were grown to 70% confluence and then switched to serum-free media. A cell suspension (1×10⁵ cells/ml, 150 μl) was added to the upper well in serum-free media containing 0.1% BSA (Sigma). Concentrations of PDGF-BB from 0.5 to 20 ng/ml were added to the bottom chambers. For all experiments, membranes were covered with type I collagen (trans-migration through type I collagen). The chambers were incubated at 37°C in a CO2 incubator for 18 hrs, and fixed in 4% formaldehyde. The non- invaded cells were removed from the upper wells and the invaded cells were stained with 0.2 % Crystal Violet solution in 7% ethanol. Cells from 8–10 randomly chosen high-power fields (magnification X20) on the lower surface of the filter were counted¹².

DNA transfection and luciferase assays

Smooth muscle cells (2×10⁵) were plated in each well of a six-well plate, incubated overnight at 37°C, media was changed to serum-free media, and then transiently transfected with plasmid DNA, either SM-actin, SM22 or p21 promoter constructs (2 μg), using the MIRUS DNA transfection method (Fisher Scientific; Waltham, Massachusetts, USA) and then co-transfected with FLAG-ZBP-89. The Renilla plasmid was co-transfected to serve as an internal control for transfection efficiency. Cells were harvested 48 h after transfection. Cell lysates were prepared via the freeze-thaw method. Dual-luciferase assays were performed according to the established protocol of Promega. All transfections were performed in triplicate with at least two different DNA preparations for each plasmid.

ChIP Analysis:

Quantitative chromatin immunoprecipitation assays (ChIP) were performed as described previously^{20, 43–46}. For each in vivo experiment, fifteen AAA samples were pooled into three sets of five samples each and ChIP analysis was performed. For qPCR, 2 μl out of the 20 μl of extracted DNA was used in 50 cycles of amplification in 3 steps: 95°C for 15 s, 55°C for 30 s, and 68°C for 45 sec. At the end of the amplification cycles, dissociation curves were determined to rule out signal from primer dimers and other nonspecific dsDNA species. Data was normalized to IgG immunoprecipitated DNA levels. Experiments were carried out in triplicate and one representative experiment was shown. The size of the PCR products was confirmed on a 2% agarose gel stained with ethidium bromide. Experiments were carried out in triplicate and one representative experiment was shown. Antibodies include polycolonal ZFP148 (Abcam). Real-time PCR primers were designed as previously described for SMA, SM22α, cFOS, and SM-MHC^{20, 43–4}. For p21 and BAK promoter assays, PCR primers were previously identified and 2 μl out of the 20 μl of extracted DNA was used in 50 cycles of amplification in 3 steps: 95°C for 15 s, 55°C for 30 s, and 68°C for 45 sec^{47, 49, 50} At the end of the amplification cycles, dissociation curves were determined to rule out signal from primer dimers and other nonspecific dsDNA species. Data was normalized to IgG immunoprecipitated DNA levels. The size of the PCR products was confirmed on a 2% agarose gel stained with ethidium bromide.

Statistical Methods

Statistical analysis of aortic diameters was performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Maximal aortic dilation (%) was calculated as [maximal aortic diameter – internal control diameter] ÷ internal control diameter * 100%. The internal control was a small segment of normal proximal abdominal aorta just distal to the renal arteries that was above the proximal extent of the AAA. This section was not perfused with elastase, but it was susceptible to blood pressure changes from volume loss during the harvest as well as expected growth with the animal over time. Values are reported as mean ± standard error of the mean. Aortic dilation between groups was compared using a student’s t-test for 2 sample groups or ANOVA for 3 or more sample groups. Post hoc Tukey correction was applied to determine the significance of individual comparisons with α=0.052. To compare two groups in other samples, a Student’s t-test was used. Paired data were analyzed by paired student’s t test. Differences between 2 or more groups versus a control group were analyzed with One-way ANOVA plus Bonferroni correction for multiple comparisons. Non-parametric data were analyzed by Mann-Whitney U test.

Results

ZNF148 is up-regulated in human and murine aneurysms and is located in smooth muscle α-actin+ cells

ZFP148 has previously been shown to be a key regulator of atherosclerosis in coordination with p53³⁰; however, no studies have investigated the role of ZFP148 in VSMC phenotypic modulation or aortic aneurysm disease³⁰. We also reasoned that since ZFP148 is closely related to the transcription factor Sp1 and Sp1 is also known to play a role in “phenotypic switching” of SMCs⁵¹, ZFP148 might also be important for this process. Therefore, we first sought to determine if ZNF148 mRNA expression increases during AAA formation in humans. MRNA Expression of ZNF148 in human AAA was found to be three-fold higher than healthy donor aortas as measured via qPCR analysis [Fig. 1A]. Next, ZNF148 protein level was analyzed via immuno-histochemical staining of human AAA samples versus control health donors. ZNF148 protein staining was markedly increased in human aneurysm tissue versus controls via immuno-histochemistry [Fig. 1B].

Figure 1: ZNF148 mRNA is elevated in human AAAs and localizes to vascular smooth muscle cells.

A) qPCR of ZNF148 mRNA in human AAA tissue(n=10) versus healthy donor abdominal aorta (n=10). * indicates significant expression over controls [p=0.0034]. B) Immuno-histochemical staining of ZNF148 in control (n=10) versus AAA (n=10). C) Confocal immuno-histochemistry performed on human AAA tissue stained with ACTA2(green), ZNF148(red), and cell nuclei(blue). D) qPCR of ZNF148 mRNA in human AAA male tissue(n=10) versus female AAA (n=6) versus controls (n=10). * indicates significant expression over controls by one-way ANOVA. E) Immuno-histochemical staining of ZNF148 in female (n=6) versus male AAA tissue (n=10).

Following these observations, we wanted to determine whether there could exist a gender difference between ZNF148 protein or mRNA levels in male versus female AAAs. MRNA expression of ZNF148 in male AAA was significantly increased compared to female AAA and compared to normal aorta controls [Fig. 1D] as was ZNF148 protein levels via immuno-histochemical staining [Fig. 1E].

Since ZNF148 staining appeared strong in different areas of the human AAA, we next performed confocal immunofluorescence to examine possible cell types ZNF148 protein could be present. Confocal immunofluorescence of male AAA samples (n=10) demonstrated that ZNF148 (red) and ACTA2 (green) were found to be localized in the same cells within human tissue [Fig. 1C]. We also performed confocal immunofluorescence in human and mouse samples with macrophages, B-cells, and T-cells present and found that ZNF148 also appears to be present within macrophages in AAAs [Supplemental Figures I, II]³⁰. These data suggest that ZNF148 may be relevant in the pathogenesis of human aneurysm formation and laid the groundwork for investigating the role of ZNF148 in smooth muscle cells during aneurysm formation.

Following our preliminary studies in human samples, we examined ZFP148 mRNA and protein expression in the elastase perfusion AAA mouse model. Aortic dilation in WT mice was significantly increased following elastase perfusion as compared to saline perfused controls [Supplemental Fig. IIIA). ²⁰. At baseline, murine aortas have relatively low levels of ZFP148 mRNA expression [Supplemental Fig. IIIB, day 0 time point]. However, ZFP148 mRNA expression significantly increased at 3, 7, 14, 21 and 28 days following elastase perfused aortas, but not in saline perfused aortas by qPCR. Interestingly, ZFP148 mRNA expression decreases after day 14, similar to the decreases seen in aortic dilation after day 14. Furthermore, ZFP148 protein staining increased following elastase perfusion in WT mice by immuno-histochemistry using an antibody specific for ZFP148 (Abcam, ab69933). [Supplemental Fig. IIIC and D]. These data suggest that ZFP148 is activated during experimental AAA formation.

ZFP148 deletion results in attenuated Aneurysm Formation

As ZFP148 was present in both human and murine AAAs and appears to be present in VSMCs and multiple inflammatory cells, these finding led us to hypothesize that ZFP148 could play an important role in AAA formation. To test this hypothesis, we created unique transgenic ZFP148 conditional knockout mice, since conventional ZFP148 knock-out mice were embryonic lethal at E9.5³³. These mice were generated as tamoxifen inducible ZFP148 flx/flx²⁷ ERT Cre+ mice and ZFP148 flx/flx ERT Cre- mice (littermate control). These inducible ZFP148 deficient mice received a series of 10 tamoxifen injections to induce ZFP148 loss in all cell types. After a two week washout period to allow Tamoxifen to be cleared, porcine pancreatic elastase was perfused into the infrarenal aorta, mice were recovered and were harvested after 14 days [Fig. 2A]. ZFP148 deficiency led to a significant reduction in maximal aortic dilation compared to control mice [ZFP148 ERTCre+: 60.51+/−15.484% n=12 vs. ZFP148 ERTCre- flx/flx: 100.8+/−13.326% n=11, p<0.0023, Fig. 2B, Supplemental Figure IV]. Given the significant difference in aortic dilation between ZFP148 ERTCre+ mice and their controls, we next sought to examine whether there were key differences in inflammatory cells, elastin/collagen deposition, proliferation or apoptosis by immuno-histochemical analysis. Vernhoff’s Van Gieson staining demonstrated significantly less elastin depletion [Fig 2C]. Moreover, ZFP148 deletion resulted in decreased macrophage, T-cell, B-cell, and neutrophil staining compared with WT controls [Supplementary Fig. V:A and B]. Interestingly, proliferation as measured by Ki67, was not changed in ZFP148 knockout, but apoptosis as measured by cleaved Caspase 3 protein staining levels was significantly decreased in ZFP148 knockouts compared to controls at 14 days [Figure 2C]. The preceding data suggests that ZFP148 is a regulator for macrophage and neutrophil recruitment. More importantly, the data also suggest that ZFP148 might regulate apoptosis during AAA formation.

Figure 2: ZFP148 ERT² Cre deletion attenuates aneurysm formation.

A) Model depicting the timeline of tamoxifen injections followed by recovery and subsequent elastase perfusion for the ZFP148 ERTCre +/− mice. B) Maximal % aortic dilation is shown for the two groups of mice at day 14 [ZFP148 Δ/Δ ERTCre+: 60.51+/−15.484% n=12 vs. ZFP148 flx/flx ERTCre-: 100.8+/−13.326% n=11, p=0.0021 by student’s t-test, Fig. 2D]. C) Elastin fibers shown with Verhoeff-van Gieson (VVG) staining. Proliferation and apoptosis were assessed using Ki-67 and cleaved Caspase 3 staining respectively. Statistical differences measured by student’s t-test.

Smooth muscle specific deletion of ZFP148 attenuates aneurysm formation

Following the total conditional knock-out studies of ZFP148 in AAA formation, we next sought to determine whether ZFP148 could be important in VSMCs during AAA formation. To investigate the possible roles of ZFP148 in SMCs, smooth muscle specific myosin heavy chain (MYH) Cre mice⁵² were bred with ZFP148 flx/flx mice to produce novel ZFP148 flx/flx MYHCre+, ZFP148 flx/wt MYHCre+, and ZFP148 wt/wt MYHCre+ mice [Fig 3A]. Smooth muscle specific conditional deletion of 1 or 2 alleles of ZFP148 resulted in a step-wise (dose dependent) protection from AAA formation as compared to control tamoxifen treated mice [ZFP148 Δ/ΔMYHCre+: 63.49 +/−15.550% n=11 vs. ZFP148 Δ/wt MYHCre+ 95.53+/− 16.84 n=9 vs. ZFP148 wt/wt MYHCre+: 113.6+/−18.45 n=15; Fig 3B, Supplemental Fig. VI]. Following our analysis of the aortic diameter, we also sought to determine whether we saw decreased macrophage, neutrophil and cleaved Caspase-3 protein staining. Verheoff Van Geison staining demonstrated that elastin depletion was reduced in heterozygous ZFP148 Δwt MYHCre+ mice and homozygous ZFP148 Δ/Δ MYHCre+ mice versus WT controls [Fig. 3C and 3D]. Similarly, there was evidence of decreased numbers of macrophage and neutrophil infiltration in the VSMC selective knockout animals as compared to WT animals [Supplemental Fig VII]. Smooth muscle cell deletion of ZFP148 resulted in significantly less cleaved Caspase 3 staining[Fig. 3C and 3D]. Interestingly, SMC specific deletion of ZFP148 resulted in increased Ki-67 staining, indicating that ZFP148 may be important for proliferation in the SMC specific knock-outs [Fig. 3C and D]

Figure 3: Vascular smooth muscle specific deletion of ZFP148 results in attenuated aneurysm formation.

A) Model depicting the timeline of tamoxifen injections followed by recovery and subsequent elastase perfusion for the ZFP148 MYHCre+/− mice. B) Maximal % aortic dilation is shown for the three groups of mice at day 14. Sample images are shown [ZFP148 Δ/Δ MYHCre+: 63.49+−15.550% n=11 vs. ZFP148 Δ/wt MYHCre+: 95.53+/−16.84% n=9 vs. ZFP148 wt/wt MYHCre+:113.6+/−18.45 n=15, statistics measured by one-way ANOVA]. C) Elastin fibers shown with Verhoeff-van Gieson (VVG) staining. Proliferation and apoptosis were visualized by Ki-67 and cleaved Caspase 3 staining respectively. Quantification of staining by student t-test for VVG, one-way ANOVA for KI67 staining, and student’s t-test for cleaved Caspase 3.

ZFP148 binding to the Bak and p21 promoter during AAA formation

ZFP148 protein had been found to bind directly to the p21 promoter to promote p21 promoter activation during cancer²⁶; therefore, although we saw conflicting results with proliferation in our conditional and SMC specific knock-out mice, we sought to determine whether ZFP148 could also alter proliferation during AAA formation^{25, 26}. We began by perform confocal analysis of Ki67 coupled with staining for SM-actin and a fibroblast marker, S100A4, in VSMC ZFP148 KO versus control mice and quantifying the results to perform a more in-depth analysis of proliferation during AAA formation [Supplemental Fig VIIIA and B]. Confocal immunofluorescence coupled with single cell quantification using imageJ demonstrated that the number of Ki67+/SM-actin+ cells was increased in the ZFP148 SMC KO mice.

We next moved to mouse smooth muscle cells where we compared transfection of a siZFP148 with adenoviral infection of ZFP148 followed by proliferation analysis using the reagent WST1⁴⁷. Incubation with the WST1 proliferation reagent demonstrated that elimination of ZFP148 in smooth muscle cells increases their proliferation [Supplemental Fig VIIIC]. Finally, we also transfected siRNAs to ZFP148 or control and then treated with increasing concentrations of PDGF-BB and assessed migration across a transwell [Supplemental Fig. VIIID.]¹². siRNA elimination decreased migration across a transwell for all treatments with PDGF-BB.

Returning to the possibility that ZFP148 regulation of proliferation could be associated with its known interaction with the cell cycle regulator, p21, we examined the levels of p21 protein expression by immunohistochemistry [Fig. 4A] and realized that we needed a more in depth confocal-based analysis to more clearly define the role of p21 gene activation and ZFP148 in AAA formation. Therefore, we performed confocal analysis of p21 in ZFP148 smooth muscle cell conditional knock-out versus littermate control mice to demonstrate that p21 declines in both SMCs (ACTA2, green) and fibroblasts (S100A4, red) during AAA formation [Fig 4B]. We performed siRNA analysis followed by treatment with elastase and saw that p21 gene transcription is normally activated following treatment with elastase [Fig. 4C]. However, treatment with an siRNA to ZFP148 resulted in a significant decline in p21 mRNA gene transcription in VSMCs treated with elastase. Trasient transfection analyses in smooth muscle cells of p21 promoter constructs demonstrates that co-transfection of a cDNA for ZFP148 coupled with the full p21 promoter construct shows increased activation while a deletion mutant or a site-directed mutation of the GC boxes in the p21 promoter decreased activation to baseline [Fig. 4D and promoter schematic]. Since there is also significant literature to suggest that ZFP148 directly activates the p21 promoter in other cell types^{25, 26}, we sought to determine whether ZFP148 could also be activating the p21 promoter during AAA formation. In vivo ChIP analyses of saline versus elastase perfused C57/B6 mice demonstrated that ZFP148 binds to the p21 promoter during AAA formation [Fig. 4E]⁴⁷.

Figure 4: ZFP148 binds the p21 promoter during AAA formation.

A) Immuno-histochemical staining of ZFP148 Δ/Δ MYHCre and ZFP148 wt/wt MYHCre+ mice for p21 protein levels (n=10/group). B) Confocal immuno-histochemistry performed on mouse AAA tissue (n=10/group) stained with ACTA2(smooth muscle, green), p21 (proliferation, yellow), S100A4 (Fibroblasts, red), and DAPI(Blue). C) qPCR analysis of VSMC cells treated with siRNAs to ZFP148 or a control followed by treatment with elastase. Cells were harvested and underwent mRNA extraction and qPCR analysis for p21 expression. Results are the average of three independent experiments performed in triplicate with * indicating significant change over elastase treated samples with a p-value< 0.05. D) p21 promoter assays plus or minus ZFP148 in mouse VSMCs. Results are the average of three independent experiments performed in triplicate. E) 14 day elastase and saline perfused C57/B6 mice underwent ChIP analysis for ZFP148 binding in vivo during aneurysm formation¹⁴. Aortas were ground and subsequently underwent formalin crosslinking and sonication. Following sonication and pre-clearage, the homogenate underwent immunoprecipitation for either ZFP148 or IgG. The precipitate was then washed and the crosslinking was reversed and proteins denatured. RT-PCR was then performed for the p21 promoter. Results are the average of three independent pooled experiments of 15 animals each performed in triplicate. *indicates significant binding over controls with a p-value <0.05 by student’s t-test.

Since there was decreased cleaved Caspase 3 staining in both the conditional and the VSMC specific ZFP148 knock-out mice during AAA formation, we sought to determine whether ZFP148 could be responsible for changes in apoptosis during AAA formation. To test this, we examined VSMC ZFP148 conditional KO mice by confocal microscopy for BAK (yellow), SM-actin (ACTA2, green) and fibroblasts (S100A4, red) coupled with cell counting using imageJ [Figs 5A and 5B]^{29, 50}. Cell analyses coupled with confocal imaging suggest that VSMC targeted ZFP148 elimination decreased the number of BAK+ smooth muscle cells in the AAA. We next treated isolated abdominal aortic mouse VSMCs with either ZFP148 or a control siRNA and then treated with either elastase or saline to investigate the mechanism of our aneurysm models in vitro. Following treatment, we extracted mRNA and performed qPCR on the samples to determine whether knock-down of ZFP148 in VSMCs using siRNA modulates BAK expression. qPCR analysis of the cells demonstrated that knock-down of ZFP148 in SMCs resulted in an inhibition of BAK [Fig. 5A]. These results mimic data found in cancer cell types²⁹. Following this observation, we followed this analysis with in vivo ChIPs analysis to determine whether ZFP148 was able to bind to the BAK promoter^{29, 50} [Fig 5C]. In vivo ChIP analysis demonstrated that ZFP148 does bind to the BAK promoter during AAA formation. Finally, siRNA knock-down of ZFP148 in VSMCs resulted in an activation of p53 mRNA expression. These data demonstrate that ZFP148 modulates apoptosis in part by activation of BAK and inhibits proliferation in part by binding to the p21 promoter.

Figure 5: ZFP148 activates the Bak promoter during AAA formation.

A) Confocal immuno-histochemistry performed on mouse AAA tissue(n=10/group) stained with ACTA2(smooth muscle, green), BAK (apoptosis, yellow), S100A4 (Fibroblasts, red), and DAPI(Blue). B) Immuno-histochemical staining of ZFP148 Δ/Δ MYHCre and ZFP148 wt/wt MYHCre+ mice for Bak protein levels (n=10/group). C) Quantification of counting of individual smooth muscle or fibroblast cells that also stain for Ki67. D) qPCR analysis of SMC cells treated with siRNAs to ZFP148 or a control followed by treatment with elastase. Cells were harvested and underwent mRNA extraction and qPCR analysis for BAK mRNA levels. Results are the average of three independent experiments performed in triplicate with * indicating significant change over elastase treated samples with a p-value< 0.05. E) 14 day elastase and saline perfused C57/B6 mice underwent ChIP analysis for ZFP148 binding in vivo during aneurysm formation¹⁴. Aortas were ground and subsequently underwent formalin crosslinking and sonication. Following sonication and pre-clearage, the homogenate underwent immunoprecipitation for either ZFP148 or IgG. The precipitate was then washed and the crosslinking was reversed and proteins denatured. RT-PCR was then performed for BAK promoter. Results are the average of three independent pooled experiments of 10 animals each performed in triplicate. Statistical analysis performed by student’s t-test. F) qPCR analysis of SMC cells treated with siRNAs to ZFP148 or a control followed by treatment with elastase. Cells were harvested and underwent mRNA extraction and qPCR analysis for Trp53.

ZFP148 knockdown inhibits down-regulation of SMC marker genes

Since ZFP148 was localized to smooth muscle cells in both Human and mouse AAA samples in our initial findings, we hypothesized that another mechanism of protection from AAA development in the global and SMC specific knock-out of ZFP148 was via modulation of VSMCs. To test this, we extracted RNA and performed qPCR analysis of SMC differentiation marker genes in global ZFP148 knockout mice versus WT control mice following elastase perfusion. SM-α were altered in the global ZFP148 knockout mice [Supplemental Fig IXA] and SM-actin protein expression was increased in global and conditional ZFP148/ZBP89 KO mice as compared with WT controls [Supplemental Fig. IXB and IXC]. To further elucidate the mechanisms for this effect, we tested if siRNA induced suppression [Supplemental Fig. X] of ZFP148 attenuated repression of SMC gene expression in response to treatment of primary cultures of abdominal aortic VSMC with elastase [1 U/mL]²⁰. Results showed that elastase-induced repression of SM α-actin induced repression was attenuated by ZFP148 knockdown [ Fig. 6A, C, E]. Furthermore, transient transfection assays of increasing amounts of ZFP148 with the SM α-actin or SM22α promoter demonstrate decreased reporter gene activity. Interestingly, higher doses of ZFP148 repression were inhibited by the presence of the G/C Repressor element, a DNA response element known to bind pELK-1 and KLF4[ Fig. 6G]⁴³. To determine if ZFP148 might directly regulate suppression of SMC marker genes in vivo, we performed chromatin immunoprecipitation (ChIP) assays on chromatin extracted from aortas from WT mice 14 days following elastase perfusion[Fig 6B,D,F]^{20, 43}. We observed enriched binding of ZFP148 to the promoters of multiple smooth muscle marker genes, including SM α-actin[Fig. 6B], SM22α [Fig. 6E], and SM-MHC[ Fig 6F.]. In contrast, there was not enriched ZFP148 binding to the C-FOS promoter following elastase perfusion [Supplemental Fig. XI].

Figure 6: siRNA Knock-down of ZFP148 modulates SMC genes following elastase treatment and binds SMC marker genes in vivo following aneurysm formation.

A,C,E) Mouse abdominal aortas were isolated, the adventitial and endothelial layer stripped and the abdominal cells were placed in culture. Cells were cultured until passage 6 and smooth muscle marker genes were verified at passage six (Data not shown). From these cultures, cells were plated at 1×10⁵ cells per well and treated with siZFP148 or control, scrambled siRNA in serum free media^{5, 12}. 24 hours following transfection cells were treated with elastase for 24 hours and then harvested, RNA extracted, and qPCR performed. * indicates significant up-regulation of vehicle treated cells in response to siZFP148 expression as compared with control treated siRNA by student’s t-test. Results are the average of three independent experiments performed in triplicate. B,D,E) 14 day elastase and saline perfused C57/B6 mice underwent ChIP analysis for ZFP148 binding in vivo during aneurysm formation¹⁴. Aortas were ground and subsequently underwent formalin crosslinking and sonication. Following sonication and pre-clearage, the homogenate underwent immunoprecipitation for either ZFP148 or IgG. The precipitate was then washed and the crosslinking was reversed and proteins denatured. RT-PCR was then performed for SMA, SM-MHC and SM22. Results are the average of three independent pooled experiments of 10 animals each performed in triplicate. Statistical analysis measured by student’s t-test. F) SMC promoter gene transfection in smooth muscle cells with increasing concentrations of ZFP148.

Following this observation, we sought to determine the possible location and mechanism of binding for ZFP148 during AAA formation. Transient transfection analyses were performed using an SM22-LacZ promoter construct coupled with various transcription factors to determine whether certain factors could be binding in concert with ZFP148 (namely, Sp1, Sp3, YY1, NF-1, KLF2, KLF5 and NRF2) coupled with in vivo ChIP analyses(data not shown) to determine if there are factors that couple with ZFP148 to promote repression of VSMC marker genes following release by KLF4. Transient transfection suggested that NF-1 and ZFP148 could act together to synergistically inhibit VSMC marker gene expression [Supplemental Fig. XII] and that these effects appeared to be mediated through the NF-1 binding site within the SM22 promoter. To further elucidate the mechanisms for this effect, we tested if shRNA induced suppression [Supplemental Fig. XIII] of NF-1 attenuated repression of VSMC gene expression in response to treatment of primary cultures of abdominal aortic VSMC with elastase [1 U/mL]²⁰ or PDGF-BB [10 ng/mL]⁴³. Results showed that elastase or PDGF-BB-induced repression of SM α-actin and SM22α was attenuated with NF-1 knockdown. Using these cells, we separately performed ChIP analysis and determined that ZFP148 was still capable of binding the SM α-actin or SM22α promoter following treatment with an shRNA to NF-1. These shRNA elimination experiments suggest that NF-1 is required for ZFP148 to bind to DNA [ Supplemental Fig. XIIIA and XIIIB]. Furthermore, elimination of NF-1 also decreased HDAC5 binding following AAA formation. Conversely, NF-1 inhibition appears to prevent de-acetylation of chromatin and appears to result in the continued presence of BRG1, an ATPase that is part of the SWI/SWF chromatin remodeling complex, on the SMC promoters⁵³. Collectively, these data suggest ZFP148 plays a role in aneurysm formation via SMC modulation.

ZFP148 deletion attenuates aneurysm formation following Angiotensin II treatment

Since the elastase perfusion model is an acute surgical model of AAA, we sought to determine if ZFP148 could also attenuate aneurysm formation in two separate Angiotensin II models of aneurysm formation. To determine if ZFP148 is protective of aneurysm formation we used: 1) ZFP148 flx/flx MYHCre+ mice bred on an ApoE−/− background infused with Angiotensin II for 28 days³⁸ [Fig. 7A]; and 2) ZFP148 flx/flx ERTCre+ mice infused with Angiotensin II with a series of TGFβ jections [Supplemental Fig. XV]⁴². We used a series of TGFβ Mallat et al., and our laboratory have demonstrated that giving C57/B6 mice a series of TGFβ ApoE−/− background^{20, 42}. Smooth muscle specific knock-out of ZFP148 was protective of mouse survival over the 28 day course of aneurysm formation following Angiotensin II pump insertion [Fig. 7B, Chi²=5.797 p-value=0.0161 by log-rank test]. The smooth muscle specific ZFP148 conditional deletion mice demonstrated a decreased aneurysm incidence [Fig. 7C] and decreased aneurysm severity [Fig. 7D]^{40, 54}. Finally, VVG, and cleaved Caspase-3 staining also declined in the Angiotensin II SMC specific ZFP148 conditional deletion mice [Supplemental Fig. IVX]. Kaplan-Meier curves of ZFP148 flx/flx ERTCre+ deleted mice demonstrated that ZFP148 protects from death due to aneurysm rupture in a separate Angiotensin II model of AAA using C57B6 mice treated with a combination of Angiotensin II and TGFβ [Supplemental Fig. XVB, Chi²=4.683, p-value= .0305 by log-rank test]. These mice demonstrated a decreased incidence of aneurysm formation in the global conditional deletion versus control littermates. [Supplemental Fig. XV]. Finally, VVG and cleaved Caspase 3 staining were significantly decreased while Ki67 demonstrated a down-ward trend. These data further demonstrate the critical importance of smooth muscle derived ZFP148 in two additional models of aneurysm formation.

Figure 7: VSMC ZFP148 knock-out attenuates aneurysm formation following Angiotensin II infusion.

A) Model depicting the timeline of tamoxifen injections followed by recovery and subsequent Angiotensin II pump insertion for ZFP148 wt/wt, Δ/wt, and Δ/Δ mice. B) Kaplan-meier survival curves of ZFP148 flx/flx MYH+ mice following Angiotensin II pump insertion with log-rank tests as indicated. C) Aneurysm Incidence in ZFP148 flx/flx MYH Cre +/− mice. D) Assessment of aneurysm severity score in ZFP148 flx/flx MYH Cre+ mice. Severity scores were based on those developed by Daugherty et al.

Discussion

Through these studies, we demonstrated that ZFP148 is a regulator of AAA formation. We demonstrated that deletion of ZFP148 results in decreased AAA formation in both a conditional and VSMC specific mouse AAA model. ZFP148 conditional and SMC specific deletion also increased mouse survival in two separate Angiotensin II models of AAA formation. We demonstrated several possible mechanisms of ZFP148 during AAA formation: 1) ZFP148 attenuates SMC marker gene expression and binds to SMC marker genes in vivo during aneurysm formation; 2) ZFP148 binds to and activates the BAK promoter during AAA formation; and 3) ZFP148 binds to the p21 promoter. The present studies demonstrate that ZFP148 contributes to AAA formation and does so at least in part by promoting phenotypic switching of SMC through direct binding to SMC marker genes. These results are somewhat paradoxical, since the dogma is that transition of VSMC to a more proliferative, migratory, synthetic state, would be beneficial in inhibiting AAA formation and/or progression. One intriguing possibility is that ZFP148 regulates transition of differentiated medial VSMC into phenotypes that may exacerbate rather than attenuate AAA pathogenesis. In this regard, it is of interest that Shankman et al¹⁹., recently used SMC lineage tracing methods to demonstrate that a subset of VSMC transition to cells that express several macrophage marker genes in the context of experimental atherosclerosis. Therefore, it is interesting to speculate that ZFP148 could exacerbate AAA pathogenesis by promoting transition of VSMC to an inflammatory macrophage-like state. It would also be of interest in the future to examine how the ZFP148 activated smooth muscle cell differs from the activated KLF4 smooth muscle cell. The Owens laboratory recently demonstrated profound differences in the effects of KLF4 and Oct4^{19, 35} during vascular disease formation and it would be of interest in the future to investigate whether ZFP148 is part of that interplay. Further studies will be required to directly test this possibility.

It will also be of interest to determine mechanisms that result in activation of ZFP148 mRNA expression in the setting of AAA. The ZFP148 promoter contains a number of conserved regulatory elements for AP-2, multiple Sp1 sites, NF-B, and Oct1 transcription factors⁵⁵. These transcription factors have been implicated previously in AAA development; therefore, it would be interesting to speculate where in the inflammatory activation pathway does ZFP148 lie in relation to these factors. A detailed analysis of ZFP148 activation and the mechanisms of inhibition of ZFP148 activation during AAA formation could represent a reasonable drug target against aneurysm formation. Second, the mechanisms responsible for controlling the different roles of ZFP148 in various cell types during vascular injury and/or disease remain unclear and, at times, contradictory. It is possible that post-translational modifications, such as differential mRNA splicing, or chemical modifications, such as phosphorylation or acetylation, regulate effects of ZFP148 in different cell types as it does for other zinc-finger transcription factors⁵⁶. ZFP148 is the second Kruppel-like zinc finger transcription factor we have shown to be important in aortic aneurysm formation and there may also be interaction between these Kruppel-like zinc-finger transcription factors during aneurysm formation as many of these factors contain Sp or Kruppel-like binding sites within their promoters⁵¹. Further studies are needed to determine if these factors regulate activation of ZFP148 in VSMC in vivo and if post-translational modifications regulate ZFP148 function like many other kruppel-like zinc-finger transcription factors.

In considering the possible limitations of this study, we are unable to ascertain the direct effect of ZFP148 on inflammatory cell infiltration without the creation of macrophage, neutrophil and T-cell specific conditional knock-out mice. Many of these conditional knock-outs are cross reactive and eliminate several cell types at any given time, thus confounding the results of these inflammatory cell conditional mouse models. Another possible limitation of this study is since ZFP148 and Sp1 are closely related, compensation could occur between ZFP148 and other Kruppel-like zinc finger transcription factors family members that could have given us our results within our mouse models. To address these limitations, we would need to perform conditional double knock-out mouse models. However, we feel these double conditional knock-outs would be beyond the scope of this study. Perhaps in future studies we will examine the possibility of compensation and the relationship between Sp1, KLF4 and ZFP148 during aortic aneurysm formation. It would be interesting to postulate whether their roles remain similar to other cell types or disease mechanisms.

In summary, the present studies provide the first evidence that ZFP148 is a regulator of AAA formation through its effects on regulating BAK and p21 transcriptional activation during AAA formation. We also demonstrate that ZFP148 plays a role in the “phenotypic switching” of VSMCs. While we can speculate that ZFP148 may work to promote a more inflammatory phenotype of VSMCs based on our current data, detailed lineage tracing analysis is needed to delineate the actual role of ZFP148 in VSMC phenotype modulation during AAA formation or atherosclerosis. These studies represent possible mechanisms for ZFP148 during AAA formation and, as demonstrated by the changes in inflammatory cells in both sets of knockout mice, ZFP148 may also have broad significance for the overall aneurysm phenotype. Future studies by our laboratory and others are needed to directly test if ZFP148 plays roles in these other cell types during aneurysm formation.

Highlights:

• ZFP148 deletion attenuates aortic aneurysm formation.

• ZFP148 activates the p21 promoter during aneurysm formation.

• ZFP148 activates the BAK promoter.

• ZFP148 and NF-1 coordinately bind to represent VSMC marker genes.

Nonstandard Abbreviation and Acronyms:

AAA

Abdominal aortic aneurysm

ACTA2

smooth muscle α-actin

ApoE

apolipoprotein E

ECM

extracellular matrix

KLF4

Kruppel-like factor 4

LGAL3

mac2

MYH11

smooth muscle cell myosin heavy chain

NF-1

neurofibromin 1

SM22α

transgelin

Trp53

p53 gene

VSMC

vascular smooth muscle cell

ZFP148

Zinc-finger protein 148

ZNF148

human Zinc-finger protein 148