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Highly Selective PPARα (Peroxisome Proliferator‐Activated Receptor α) Agonist Pemafibrate Inhibits Stent Inflammation and Restenosis Assessed by Multimodality Molecular‐Microstructural Imaging

Originally publishedhttps://doi.org/10.1161/JAHA.121.020834Journal of the American Heart Association. 2021;10:e020834

Abstract

BACKGROUND

New pharmacological approaches are needed to prevent stent restenosis. This study tested the hypothesis that pemafibrate, a novel clinical selective PPARα (peroxisome proliferator‐activated receptor α) agonist, suppresses coronary stent‐induced arterial inflammation and neointimal hyperplasia.

METHODS AND RESULTS

Yorkshire pigs randomly received either oral pemafibrate (30 mg/day; n=6) or control vehicle (n=7) for 7 days, followed by coronary arterial implantation of 3.5 × 12 mm bare metal stents (2–4 per animal; 44 stents total). On day 7, intracoronary molecular‐structural near‐infrared fluorescence and optical coherence tomography imaging was performed to assess the arterial inflammatory response, demonstrating that pemafibrate reduced stent‐induced inflammatory protease activity (near‐infrared fluorescence target‐to‐background ratio: pemafibrate, median [25th‐75th percentile]: 2.8 [2.5–3.3] versus control, 4.1 [3.3–4.3], P=0.02). At day 28, animals underwent repeat near‐infrared fluorescence–optical coherence tomography imaging and were euthanized, and coronary stent tissue molecular and histological analyses. Day 28 optical coherence tomography imaging showed that pemafibrate significantly reduced stent neointima volume (pemafibrate, 43.1 [33.7–54.1] mm3 versus control, 54.2 [41.2–81.1] mm3; P=0.03). In addition, pemafibrate suppressed day 28 stent‐induced cellular inflammation and neointima expression of the inflammatory mediators TNF‐α (tumor necrosis factor‐α) and MMP‐9 (matrix metalloproteinase 9) and enhanced the smooth muscle differentiation markers calponin and smoothelin. In vitro assays indicated that the STAT3 (signal transducer and activator of transcription 3)–myocardin axes mediated the inhibitory effects of pemafibrate on smooth muscle cell proliferation.

CONCLUSIONS

Pemafibrate reduces preclinical coronary stent inflammation and neointimal hyperplasia following bare metal stent deployment. These results motivate further trials evaluating pemafibrate as a new strategy to prevent clinical stent restenosis.

Nonstandard Abbreviations and Acronyms

BMS

bare metal stent

DES

drug‐eluting stent

NIRF

near‐infrared fluorescence

PPARα

peroxisome proliferator‐activated receptor α

SMC

smooth muscle cell

TBR

target‐to‐background ratio

Clinical Perspective

What Is New?

  • This is the first study to demonstrate that pemafibrate, a novel PPARa (peroxisome proliferator‐activated receptor α) agonist and selective PPARa modulators can reduce coronary stent‐induced vascular inflammation and neointima formation in pigs as measured by clinically translatable intravascular near‐infrared fluorescence–optical coherence tomography molecular structural imaging.

What Are the Clinical Implications?

  • Restenosis remains a significant source of morbidity following coronary and peripheral intervention, and at present, there are limited oral pharmacotherapies to prevent restenosis.

  • Study findings indicate that pemafibrate may merit evaluation as a new pharmacologic strategy to limit clinical endovascular stent restenosis.

  • Intravascular near‐infrared fluorescence–optical coherence tomography imaging may be a new translatable approach to evaluate the anti‐inflammatory actions of new drugs in vivo in coronary arteries.

Endovascular stent implantation during percutaneous coronary intervention or peripheral artery disease (PAD) intervention provides a highly effective therapy for obstructive atherosclerosis. However, stent implantation induces vascular injury and inflammation that can incite excessive neointimal hyperplasia and clinical stent restenosis, a morbid and costly condition often leading to rehospitalization and repeat intervention and potentially increased mortality.1, 2 Furthermore, despite contemporary stent technology and optimized implantation techniques, metallic stents carry an indefinite risk of late failure attributed to both fibrotic neointima formation or neoatherosclerosis, characterized by neointima containing foamy macrophages, cholesterol clefts, and rupture‐prone fibrous caps.3, 4 Therefore, new strategies to reduce stent restenosis remain urgently needed.

At present, no oral pharmacotherapies effectively prevent clinical restenosis.5 As inflammation and cellular proliferation play a crucial role in neointima formation,6, 7 therapeutics inducing both anti‐inflammatory and antiproliferative effects might potently inhibit restenosis. This study investigated a new anti‐inflammatory and antiproliferative strategy using the novel clinical oral agent pemafibrate, a highly selective PPARα (peroxisome proliferator‐activated receptor α) agonist.8 Pemafibrate represents a new class of drugs: selective PPARα modulators that favorably alter lipoprotein levels.9 In addition to its lipid modulatory capacity, PPARα activation may also exert anti‐inflammatory and antiatherogenic effects,10, 11, 12 but its effects on restenosis are undetermined.

This preclinical in vivo molecular‐structural inflammation imaging and biological investigation tested the hypothesis that selective PPARα activation with pemafibrate can suppress in vivo stent‐induced inflammation and neointima formation in pig coronary arteries as assessed by intravascular near‐infrared fluorescence (NIRF)–optical coherence tomography (OCT) hybrid imaging.13, 14, 15 To further understand the potential antirestenosis effects of pemafibrate, we investigated anti‐inflammatory and antiproliferative cellular and molecular mechanisms in cellular and organ cultures.

Methods

The authors declare that all supporting data are available within the article (and its online supplementary files).

Study Protocol

This study was approved and performed in compliance with the Massachusetts General Hospital Institutional Animal Care and Use Committee (protocol number 2012N000066). Healthy male Yorkshire pigs (2.6±0.3 months old, body weight 25–30 kg, N=13) were fed normal chow and then randomized to receive oral pemafibrate or PBS 7 days before bare metal stent (BMS) implantation (3.5 × 12 mm, Multi‐Link Vision, Abbott Vascular) into a coronary artery at a 1.3:1 stent: artery overexpansion ratio guided by intravascular ultrasound (IVUS), which was followed by dual antiplatelet therapy. Based on a previous pilot pharmacokinetics study of pemafibrate in normal pigs, a dose of approximately 1 mg/kg/day (30 mg/day) per pig was chosen for this study. BMSs were used to generate more abundant restenosis compared with drug‐eluting stents (DESs) that harbor embedded antiproliferative coatings. Pigs next underwent serial intravascular NIRF‐OCT imaging at days 7 and 28 after stent implantation (Figure S1). Following day 28 imaging, pigs were euthanized, and excision of the coronary arteries on ice occurred within 15 minutes. Arteries were then processed for histopathology, quantitative polymerase chain reaction, immunoblotting, and proteomic assays.

Intracoronary Molecular‐Structural Imaging

Intravascular NIRF‐OCT and IVUS imaging were performed in all animals on days 7 and 28 after stent implantation (Figure 1). In addition, day 7 NIRF‐OCT imaging was performed in a subgroup of animals (3 of 7 control animals and 3 of 6 pemafibrate animals). To visualize stent cathepsin proteinase inflammatory activity in vivo, intravenous ProSense VM110 (60 mg/pig, 2.1±0.1 mg/kg, PerkinElmer), a NIRF cathepsin protease activity reporter, was administered 24 hours before NIRF‐OCT imaging.13, 16, 17 ProSense VM110 (ex/em 750 nm/770 nm) is optically silent in its native state, but after lysine–lysine bond cleavage by cathepsin B, L, or S proteases, generates strong local NIR fluorescence.13, 16, 17 Data S1 provides additional details of the NIRF‐OCT imaging apparatus.

Figure 1. Study scheme and analysis protocol. Pemafibrate administration (30 mg/animal/day) was initiated at day −7 and continued until animals were harvested at day 28 (total 35‐day protocol).

On day 0, BMSs (3.5 × 12 mm) were deployed into the pig RCA and LAD coronary arteries. Arterial injury was induced by stent balloon overinflation under IVUS guidance. Intravascular imaging using IVUS and NIRF‐OCT was performed on day 7 at 24 hours after intravenous injection of ProSense VM110, a NIRF molecular imaging reporter of cathepsin protease activity. Tissues were harvested at day 28 for analysis. BMS indicates bare metal stent; IVUS, intravascular ultrasound; LAD, left anterior descending coronary artery; NIRF, near‐infrared fluorescence; OCT, optical coherence tomography; qPCR, quantitative polymerase chain reaction; and RCA, right coronary artery.

Histologic Examination, Protein Isolation and Proteomics Analysis, Quantitative Real‐Time Polymerase Chain Reaction, Ex Vivo Carotid Artery Ring Organoid Culture, and Primary Smooth Muscle Cell Culture

Detailed methods are described in Data S1.

Statistical Analysis

Data are presented as median (25th–75th percentiles) or mean±SD as appropriate. An “n” indicates the number of independent experiments or number of animals/samples. Tests with a P value < 0.05 were considered statistically significant. Pairwise group comparisons were performed using a nonparametric Mann–Whitney U test (SPSS 24, IBM Corp., Armonk, NY, and GraphPad Prism 5, Prism Software Inc, La Jolla, CA). Linear correlation between 2 parameters was performed to calculate the Spearman’s correlation coefficient (r). Factorial repeated‐measures ANOVAs as mixed models using a random intercept for each pig and an interaction between terms (treatment and level) were performed for comparisons between the pemafibrate and control groups of OCT‐derived percent stenosis in 36 levels/positions per stent (a total of 1584 OCT images analyzed across 44 stents; 24 control and 20 pemafibrate).

Results

Pemafibrate Suppresses Stent‐Induced Inflammatory Protease Activity in Pig Coronary Arteries as Assessed by NIRF‐OCT Hybrid Imaging In Vivo

After stent implantation, intravascular images revealed similar stent overexpansion at day 0 and at day 7 between groups (stent diameter : distal reference diameter ratios of 1.34–1.39; Table S1) without medial dissection, intramural hematoma, or strut malapposition. The day 7 inflammatory protease activity monitored in vivo by the NIRF imaging agent ProSense VM110, an established cathepsin protease activity reporter in arterial disease,13, 17 was captured during NIRF‐OCT imaging. The 3‐dimensional coronary artery NIRF signals were displayed on a 2‐dimensional NIRF map and quantified as the ratio of NIRF intensities in the lesion of interest to those in the nonstented regions (target‐to‐background ratio [TBR]). Pemafibrate administration reduced the NIRF cathepsin protease inflammation signal localized in the stented coronary artery at day 7 compared with stents implanted in control pigs (Figure 2A). The NIRF inflammation TBR in the pemafibrate treated group (TBR, 2.8 [2.5–3.3]; 9 stents and 3 animals) was significantly lower than the control group (TBR, 4.1 [3.3–4.3]; 9 stents and 3 animals; P=0.024; Figure 2B).

Figure 2. The effects of pemafibrate on in vivo arterial inflammatory cathepsin activity 7 days after stent implantation.

A, NIRF signals in the RCA and LAD of a representative control and pemafibrate‐treated animal. The day 7 NIRF signal intensity, reflecting inflammatory cathepsin protease activity, was lower in stents of pigs treated with pemafibrate compared with controls. The NIRF signal in nonstented areas was used as the background/reference measurement. A total of 4 representative stents are shown (left, control; right, pemafibrate [all 2‐dimensional NIRF maps equally windowed]). B, The NIRF inflammation signal measured as the TBR was significantly lower in pemafibrate‐treated animals (9 stents, 3 animals in each group). Each dot indicates 1 stent. Horizontal lines and error bars indicate medians and 25th to 75th percentiles, respectively. LAD indicates left anterior descending coronary artery; NIRF, near‐infrared fluorescence; RCA, right coronary artery; and TBR, target‐to‐background ratio. *P<0.05.

Pemafibrate Reduces Neointimal Hyperplasia in Stented Arteries

On day 28, in vivo intracoronary OCT images (44 stents and 13 animals) and Movat’s pentachrome stain of histological sections (13 stents and 13 animals) enabled the quantification of the stent neointima area (Figure 3A). Manual measurements of the coronary lumen and the stent cross‐sectional area by OCT yielded a 3‐dimensional quantitative evaluation of the neointima volume per stent in the pemafibrate and control groups. Neointima volume at day 28 was lower in the arteries of pemafibrate‐treated pigs than controls (control, 54.2 [41.2–81.1] mm3 versus pemafibrate, 43.1 [33.7–54.1] mm3; P=0.032; Figure 3B). Longitudinal analysis by comparison in 2 groups using factorial‐measures ANOVAs with mixed models further showed a significantly lower percent luminal stenosis in stents in the pemafibrate group, particularly at the distal edge of the stent (P<0.001). Analyses on a per‐stent basis by OCT showed significantly lower neointima volume, percent area, and volume stenosis in the pemafibrate group (Figure 3B, 3C, 3F, and 3G), whereas there was nonsignificant lower neointimal area and volume in the pemafibrate group on a per‐animal analysis of the OCT and histology data (Figure 3D, Figure S1a and 1b). Moreover, the NIRF protease activity TBR on day 7 correlated moderately with the day 28 neointima volume per stent (r=0.52, P=0.031), indicating that early stent‐induced NIRF inflammation may be able to predict the degree of subsequent stent neointima hyperplasia (Figure 3E). Both the day 28 OCT‐derived stent mean percent neointima area stenosis (Figure 3F) and percent neointima volume (Figure 3G) were significantly lower in stents of pemafibrate‐treated animals.

Figure 3. Assessment of pemafibrate on in‐stent neointima formation.

Representative figures of OCT imaging (day 28), histology, and 3‐dimensional representation (day 28) in the control and pemafibrate groups, respectively. A, OCT showed that pemafibrate attenuated in‐stent neointima formation. (Scale bars=1 mm.) Movat’s pentachrome demonstrated more fibrous, less mucinous (proteoglycan), and lower cell accumulation around stent struts as well as in the adventitia in the pemafibrate‐treated animals. (Scale bars=100 µm.) Representative images of 3‐dimensional neointima reconstructions in the control and pemafibrate groups. *Neointima. B, Neointima volume per stent assessed at day 28 was suppressed by pemafibrate treatment (n=20 stents, 6 animals in the control group; n=24 stents, 7 animals in the pemafibrate group). C, Longitudinal analysis OCT images showed a lower percent luminal stenosis in the pemafibrate‐treated pigs (P<0.001), particularly toward the distal stent edge. D, Longitudinal analysis of histological images showed similar findings as did the OCT analysis, with lower neointima area across the stent in the pemafibrate group; however, this trend was not statistically significant (P=0.138). Each dot represents the average histology‐measured neointimal area at a given stent distance from either control (circles) or pemafibrate (squares) subjects. E, A moderate correlation was found between the early NIRF inflammatory cathepsin protease signal on day 7 and the subsequent neointima volume on day 28 (n=17 stents). Each dot depicts 1 stent. F, Mean percentage neointima area stenosis at day 28 derived from OCT images. G, Percentage neointima volume per stent at day 28 derived from OCT images. Each dot represents 1 stent with 36 images per stent analyzed every 0.33 mm from the distal stent edge. Horizontal lines and error bars indicate medians and 25th to 75th percentiles, respectively. NIRF indicates near‐infrared fluorescence; N.S., not significant; OCT, optical coherence tomography; and Pema., pemafibrate. *P<0.05; ***P<0.001.

Effects of Pemafibrate on Body Weight, Triglyceride, and Hepatic Enzymes

At 2 to 6 hours after oral administration, the plasma pemafibrate concentration was measured at 38.2±32.8 ng/dL, whereas it was undetectable in the plasma of controls. Pemafibrate did not alter body weight during the 35‐day study. Although pemafibrate was developed as a clinical triglyceride‐lowering drug, the dose we chose did not significantly change triglyceride levels at day 28 (35 days after treatment initiation) in normolipidemic pigs (14.4±6.9 mg/dL in the controls, 12.6±7.5 mg/dL in the pemafibrate group, and 17.8±1.8 mg/dL in normal Yorkshire pigs18) who had low triglyceride concentrations at baseline. Pemafibrate did not induce hepatic enzyme elevation at day 28 (35 days of administration), including aspartate aminotransferase (25.6±15.0 U/L in the controls, 14.9±6.4 U/L in the pemafibrate group, and 48.9±10.5 U/L in normal Yorkshire pigs19) and alanine aminotransferase (39.7±7.0 IU/L in the controls, 43.3.1±11.7 IU/L in the pemafibrate group, and 50.5±4.9 IU/L in normal Yorkshire pigs19), indicating no apparent hepatic toxicity at the administered dose (Table S2).

Pemafibrate Suppresses Cell Accumulation and Proliferation in Stented Arteries

The stented coronary arteries of pemafibrate‐treated animals at day 28 demonstrated that for a given amount of neointima, there was higher α‐smooth muscle actin (SMα‐actin) area and fewer Ki‐67‐positive nuclei, a marker of cell proliferation (Figure 4A). Double immunofluorescence staining of Ki‐67 and SMα‐actin indicated that pemafibrate suppressed the accumulation of proliferating Ki‐67+ cells while increasing the presence of mature SMα‐actin+ smooth muscle cells (SMCs). Colocalization analysis (Figure 4A) further showed that most Ki‐67 cells were not SMα‐actin positive, suggesting that Ki‐67+ cells may be inflammatory cells or less‐differentiated SMCs. Fewer cells were present in the pemafibrate group on a per‐strut basis (N=504 control, N=432 pemafibrate [Figure 4B, left]; P=0.016) as well as on a per‐animal basis (N=7 control, N=6 pemafibrate, 72 stent struts analyzed per animal [Figure 4B, right]; P=0.008). Moreover, histological analysis demonstrated that pemafibrate reduced the strut‐associated inflammation, granulomatous inflammation, and vascular injury scores (all P<0.05; Figure 4C through 4E). These findings illustrate that pemafibrate suppressed stent injury‐induced cell accumulation and proliferation. At day 28, we observed a positive correlation between the day 28 stent‐induced granulomatous inflammation and neointimal area (Figure S2b; r=0.24, P=0.03). No statistically significant correlation was evident between the day 28 peri‐strut inflammation or vascular injury score and the neointimal area (Figure S2a and S2c).

Figure 4. The effects of pemafibrate on peri‐stent strut cell accumulation and proliferation.

A, Hematoxylin‐eosin and immunofluorescence staining of tissues around stent struts on day 28. Cell accumulation and the number of Ki‐67+ cells were lower in the pemafibrate group than the control group, whereas SMα‐actin expression was higher. B, Nuclear count around stent struts per 0.01 mm2 (100 × 100 µm) was significantly lower in the pemafibrate group on a per‐strut basis (left, N=504 control, N=432 pemafibrate; P=0.016 [box plots indicate medians and 25th–75th percentiles]) as well as on a per‐animal basis (right, N=7 control, N=6 pemafibrate, 72 stent struts analyzed per animal; P=0.008 [horizontal line and error bars indicate medians and 25th–75th percentiles, respectively]). C–E, Histological analysis at day 28 (n=84 sections in the control group, n=72 sections in the pemafibrate group [box plots indicate medianss and 25th–75th percentiles]) revealed lower peri‐strut inflammation, granulomatous inflammation percentage, and vascular injury scores in the pemafibrate group. (Scale bar=100 µm.) Ad indicates adventitia; I, intima; M, media; Pema., pemafibrate; S, stent; and SMα, smooth muscle α. *P<0.05, **P<0.01.

Pemafibrate Enhances the PPARα Pathway and Suppresses Inflammation and Proliferation in Experimental Stented Arteries and Human Arteries

To examine further the effects of pemafibrate on the in vivo effects of SMC biology, neointima specimens from implanted resected pig coronary artery stents were dissected for mRNA and protein expression studies. The left circumflex coronary artery without stent implantation from the same animal served as a control reference artery. As compared with reference arterial segments, the intima of stented arteries expressed higher mRNA levels of the proinflammatory cytokine TNFα (tumor necrosis factor‐α; Figure S3a). Pemafibrate treatment reduced stent TNFα expression compared with control stents. Furthermore, the induction of MMP‐9 (matrix metalloproteinase 9) in stented intima was nearly abrogated by pemafibrate. Pemafibrate further enhanced the gene expression of PPARα (consistent with prior studies in endothelial cells and osteoblasts20, 21) as well as CPT1a (carnitine palmitoyltransferase 1A), a target of the PPARα pathway, in both the neointima of stented arteries as well as in reference coronary arteries (Figure S2b). In addition, pemafibrate reduced SMC proliferation in human carotid artery culture ex vivo and promoted maturity of human coronary artery SMCs in vitro (Data S1 and Figures S4 and S5).

Pemafibrate Maintains SMC Differentiation in the Neointima of Pig Coronary Arteries

The combined use of key markers of SMC differentiation such as α‐actin, calponin, smoothelin, and myosin heavy chain helps identify the differentiated state of SMC during the development of vascular disorders such as atherosclerosis and restenosis.22, 23, 24 The availability of antibodies for such proteins in pigs, however, is limited. We therefore optimized mass spectrometry–assisted proteomics to examine the effects of pemafibrate on the differentiated state of SMC in stented arteries. The expression levels of SMC differentiation markers, such as CNN1 (calponin 1) and SMTN (smoothelin), were higher, and ACTA2 (SMα‐actin) tended to be higher in the pemafibrate‐treated arteries, whereas the expression levels of housekeeping proteins, such as GAPDH, β‐actin, and β‐tubulin, were similar in the 2 groups (Figure S6). The levels of myosin heavy chain (MYH11), indicative of fully differentiated SMCs, did not differ between groups. These results indicate that PPARα activation by pemafibrate may suppress phenotypic modulation of SMCs into the immature stage after stent implantation but do not fully restore the state of mature SMCs.

Relationship Between Serum Lipid Levels and In Vivo NIRF Inflammation

Serum lipids levels were similar between the control and pemafibrate groups at both day 0 and day 28. Serum high‐density lipoprotein cholesterol levels on day 0 and day 28 did not exhibit a significant relationship to the day 7 in vivo NIRF inflammation levels (Figure S7).

DISCUSSION

This multimodal molecular‐microstructural intravascular study demonstrates that the clinically approved potent PPARα activator pemafibrate reduces preclinical stent‐induced inflammation and restenosis in a preclinical swine model. PPARα activation in this investigation was achieved through the selective PPARα activator pemafibrate, with a potency >1000 times higher than the conventional PPARα agonist fenofibrate.9 Our work revealed that pemafibrate inhibited stent inflammation assessed by high‐resolution in vivo NIRF‐OCT inflammation imaging and suppression of mRNA neointima inflammatory mediators TNF‐α and MMP‐9 (matrix metalloproteinase 9). Pemafibrate further reduced histopathological cell inflammation and proliferation following stent‐induced vascular injury. Proteomic analyses of stented arteries, organoid cultures of pig arteries, and in vitro experiments in human primary SMCs provided additional evidence that pemafibrate suppressed SMC phenotypic modulation, proinflammatory activation, and proliferation. Overall, these results demonstrate the following: (1) that intravascular molecular‐microstructural NIRF‐OCT imaging can translationally image the anti‐inflammatory effects of new restenosis therapies in vivo and (2) that pemafibrate may merit evaluation as a new pharmacologic strategy to limit clinical endovascular stent restenosis in patients.

Stent implantation causes local endovascular injury and inflammation, leading to macrophage and SMC activation that promote in‐stent restenosis, late neoatherosclerosis, and thrombosis.6, 7, 25 In this study, intravascular NIRF‐OCT demonstrated reduced in vivo inflammatory cathepsin protease activity and stent neointimal volume in implanted bare metal coronary artery stents of pemafibrate‐treated animals. By reducing activated tissue cathepsins, such as cathepsin S, an important cysteine protease in human atherosclerosis pathobiology that promotes collagenolysis, elastolysis, and facilitates SMC migration,26, 27, 28 pemafibrate may interrupt a key step in the restenosis process. In addition to suppressing in vivo inflammatory protease activity, pemafibrate also reduced Ki‐67 cellular proliferation and promoted calponin and smoothelin markers of SMC differentiation, effectively decreasing the likelihood of neointima SMC expansion. Although the dose used in the present study does not lower plasma triglyceride levels, we used normolipidemic pigs in this study, and the study time frame was too short to evaluate longer term effects on triglyceride levels. Thus, the effects of pemafibrate on lesion development, inflammation, and SMC phenotype after stenting were likely mediated by mechanisms independent of the changes in the levels of circulating triglycerides. To further understand mechanisms of reduced neointimal volume induced by pemafibrate, additional in vitro analyses using primary cultured human SMCs demonstrated that pemafibrate fostered the mature state of SMC and limited proliferation via the STAT3 (signal transducer and activator of transcription 3) and myocardin axes. Consistent with prior studies of conventional PPARα agonists,29 and with prior pemafibrate studies in atherosclerosis,30, 31 the current results for the first time demonstrate that pemafibrate can exert anti‐inflammatory and antiproliferative effects in stented arteries, independently of its action on plasma triglycerides, and provide insight into SMC mechanisms underlying the observed pemafibrate‐driven reductions in stent restenosis.

Given the importance of inflammation in restenosis and atherosclerosis, there is increasing interest in imaging inflammation in vivo at high resolution in coronary arteries.32 To enable high‐resolution molecular imaging, our laboratories have developed intravascular NIRF imaging platforms for assessing in vivo atheroma and stent pathobiology, including inflammation and fibrin deposition.16, 33, 34 Translationally, a NIRF‐OCT system has been used in patients with coronary artery disease to detect NIR autofluorescence.35, 36 In addition to ProSense VM110, which has been evaluated clinically (NCT03286062), an analogous cathepsin reporter37 and the endothelial leakage NIRF agent indocyanine green14, 34 also appear promising for clinical NIRF imaging of coronary artery disease. The current study provides a framework for evaluating whether NIRF imaging of stent inflammation will identify subjects at higher risk for clinical restenosis and for assessing the relationship between inflammation and stent restenosis in patients receiving anti‐inflammatory pharmacotherapy.

Although the current study used BMS implantation to efficiently generate higher volumes of neointimal hyperplasia compared with antiproliferative DESs, BMSs currently remain the predominant stent type used during endovascular treatment of PAD, especially given the recent concerns about the use of paclitaxel‐coated devices.38 Moreover, restenosis after endovascular stenting for both femoropopliteal artery and below‐the‐knee PAD remains a major clinical and costly clinical problem.39, 40 Therefore, if clinically validated, pemafibrate could help improve the outcomes of patients with PAD treated with BMSs. From a coronary standpoint, although further experimental studies are needed to determine the efficacy of pemafibrate on suppressing neointimal hyperplasia in DESs, it is important to note that coronary stent restenosis remains a substantial clinical problem even in the DES era, with rates ranging between 3% to 20%.41 Coronary stent restenosis carries increased morbidity and mortality,1, 42 and treatment of refractory restenosis may require coronary artery bypass surgery or invasive intracoronary brachytherapy, which still carries a significant risk of recurrent restenosis.43 Despite decades of research44 and the potential for oral cilostazol45 or colchicine,46 no oral antirestenosis therapy is yet clinically established.

This study has certain limitations. First, we did not examine the effect of pemafibrate on suppressing DES neointima formation because the higher stent neointima formation afforded by BMSs allowed more efficient restenosis generation; of note, BMSs are routinely used in the treatment of PAD, and the use of BMSs is thus still clinically relevant. Future studies evaluating the effects of pemafibrate on the restenosis following DES implantation are needed, particularly in subjects with coronary artery disease. Second, stent implantation into the normal arteries of male juvenile swines does not fully recapitulate the pathophysiology of restenosis occurring in a milieu of atherosclerosis. Third, although various drugs including those with anti‐inflammatory capabilities47 have demonstrated an antirestenotic effect in preclinical stenting models, similar clinical antirestenotic effects have rarely been reproduced in the patients.48 Fourth, although the current study design reproduced previous large animal stent studies and indicates significant pemafibrate‐induced reductions in stent‐induced arterial inflammation and neointima volume on a per‐stent basis, as the per‐animal comparisons were not significant likely as a result of being underpowered, the NIRF‐OCT findings and the overall study findings are considered hypothesis generating. Therefore, although the current study demonstrates promising preclinical antirestenotic effects by pemafibrate, additional validation studies are needed, including those with extended time points, to determine whether pemafibrate could suppress both neointima formation and clinical restenosis rates in patients with PAD and coronary artery disease.

In conclusion, this integrative multimodal intravascular imaging and biological study demonstrates the potential for the novel PPARα selective activator pemafibrate to reduce preclinical stent in vivo inflammation, cellular proliferation, and neointimal hyperplasia and therefore may offer a new strategy to reduce clinical endovascular stent restenosis.

Sources of Funding

This study was supported by research grants from Kowa Company, Ltd, Nagoya, Japan, to Masanori Aikawa and Farouc A. Jaffer, the National Institutes of Health (R01HL126901 and R01HL149302 to Masanori Aikawa; R01HL122388, R01HL137913, and R01HL150538 to Farouc A. Jaffer; K08HL130465 to Eric A. Osborn; R01HL136431 and R01HL147095 to Elena Aikawa; and R01HL080472 to Peter Libby), American Heart Association (18CSA34080399 to Peter Libby), and the RRM Charitable Fund to Peter Libby. The near‐infrared fluorescence–optical coherence tomography system was developed in part under R01HL093717 to Guillermo J. Tearney. Kowa Company, Ltd, Nagoya, Japan, provided partial research funding support for this study but was not involved in the study design or data analysis.

Disclosures

Dr Iwata has received sponsored research from Kowa and Daiichi‐Sankyo. Dr Osborn is a consultant for Abbott Vascular and Canon, has served on the scientific advisory board and has equity interest in Dyad Medical, and has received personal fees from Opsens Medical. Dr Tearney has received sponsored research from Merck Sharp & Dohme, VivoLight, and Canon; has received royalties and catheter components from Terumo; and has been a consultant for Samsung. Dr Tearney has a financial/fiduciary interest and consults for SpectraWAVE, a company developing an optical coherence tomography–near‐infrared fluorescence intracoronary imaging system and catheter. His financial/fiduciary interest was reviewed and is managed by the Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. Dr Jaffer has received sponsored research from Kowa, Canon, Siemens, Teleflex, and Shockwave and is a consultant for Boston Scientific, Abbott Vascular, Siemens, Magenta Medical, Asahi Intecc, and IMDS. Dr Jaffer has an equity interest in Intravascular Imaging, Inc. and DurVena. Massachusetts General Hospital has a patent licensing arrangement with Terumo, Canon, and SpectraWAVE; Dr Tearney and Dr Jaffer (Terumo, Canon, SpectraWAVE) have the right to receive royalties. Dr Aikawa has received sponsored research from Kowa, Pfizer, and Sanofi. Dr Libby has been an unpaid consultant to or is involved in clinical trials for Amgen, AstraZeneca, Esperion Therapeutics, Ionis Pharmaceuticals, Kowa Pharmaceuticals, Novartis, Pfizer, Sanofi‐Regeneron, and XBiotech, Inc. and has been a member of scientific advisory boards for Amgen, Athera Biotechnologies, Corvidia Therapeutics, DalCor Pharmaceuticals, IFM Therapeutics, Kowa Pharmaceuticals, Olatec Therapeutics, Medimmune, and Novartis, and his laboratory has received research funding in the past 2 years from Novartis.

Acknowledgments

We thank Brett Pieper, Jung Choi, Tan H. Pham, Jennifer Wen, and Anna Heewoo Ha for their technical assistance.

Footnotes

* Correspondence to: Farouc A. Jaffer, MD, PhD, Division of Cardiology, Massachusetts General Hospital, Simches 3206, 185 Cambridge Street, Boston, MA 02114. E‐mail:
and
Masanori Aikawa, MD, PhD, Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women's Hospital, 3 Blackfan St, CLSB, Floor 17, Boston, MA 02115. E‐mail:

*H. Iwata, E.A. Osborn, and G.J. Ughi are co‐first authors.

G.J. Tearney, M. Aikawa, and F.A. Jaffer share senior authorship.

Supplementary Material for this article is available at https://www.ahajournals.org/doi/suppl/10.1161/JAHA.121.020834

For Sources of Funding and Disclosures, see page 10.

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