A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

Hiroshi Kawanishi; Koji Ohashi; Hayato Ogawa; Naoya Otaka; Tomonobu Takikawa; Lixin Fang; Yuta Ozaki; Mikito Takefuji; Toyoaki Murohara; Noriyuki Ouchi

doi:10.1371/journal.pone.0235362

. 2020 Jun 25;15(6):e0235362. doi: 10.1371/journal.pone.0235362

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

Hiroshi Kawanishi ¹, Koji Ohashi ^2,^*, Hayato Ogawa ¹, Naoya Otaka ¹, Tomonobu Takikawa ¹, Lixin Fang ¹, Yuta Ozaki ¹, Mikito Takefuji ¹, Toyoaki Murohara ¹, Noriyuki Ouchi ^2,^*

Editor: Masuko Ushio-Fukai³

PMCID: PMC7316279 PMID: 32584895

Abstract

Objective

Cardiovascular disease is a leading cause of death worldwide. Obesity-related metabolic disorders including dyslipidemia cause impaired collateralization under ischemic conditions, thereby resulting in exacerbated cardiovascular dysfunction. Pemafibrate is a novel selective PPARα modulator, which has been reported to improve atherogenic dyslipidemia, in particular, hypertriglyceridemia and low HDL-cholesterol. Here, we investigated whether pemafibrate modulates the revascularization process in a mouse model of hindlimb ischemia.

Methods and results

Male wild-type (WT) mice were randomly assigned to two groups, normal diet or pemafibrate admixture diet from the ages of 6 weeks. After 4 weeks, mice were subjected to unilateral hindlimb surgery to remove the left femoral artery and vein. Pemafibrate treatment enhanced blood flow recovery and capillary formation in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of endothelial nitric oxide synthase (eNOS). Treatment of cultured endothelial cells with pemafibrate resulted in increased network formation and migratory activity, which were blocked by pretreatment with the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME). Pemafibrate treatment also increased plasma levels of the PPARα-regulated gene, fibroblast growth factor (FGF) 21 in WT mice. Systemic administration of adenoviral vectors expressing FGF21 (Ad-FGF21) to WT mice enhanced blood flow recovery, capillary density and eNOS phosphorylation in ischemic limbs. Treatment of cultured endothelial cells with FGF21 protein led to increases in endothelial cell network formation and migration, which were canceled by pretreatment with L-NAME. Furthermore, administration of pemafibrate or Ad-FGF21 had no effects on blood flow in ischemic limbs in eNOS-deficient mice.

Conclusion

These data suggest that pemafibrate can promote revascularization in response to ischemia, at least in part, through direct and FGF21-mediated modulation of endothelial cell function. Thus, pemafibrate could be a potentially beneficial drug for ischemic vascular disease.

Introduction

Cardiovascular disease is a major cause of death worldwide [1]. Obesity-related metabolic disorders including type 2 diabetes and dyslipidemia contribute to impaired collateralization and vascular insufficiency under ischemic conditions, thereby leading to exacerbation of cardiac dysfunction and tissue injury [2–4]. Thus, the enhancement of collateral vessel development can be a promising therapeutic target of cardiovascular diseases.

Peroxisome proliferator-activated receptor (PPAR) α is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors, and has an important effect on lipid and lipoprotein metabolism [5]. PPARα agonists decrease plasma triglyceride levels and increase plasma high density lipoprotein (HDL)-cholesterol levels. In addition, PPARα agonists have various roles in regulation of cardiovascular homeostasis such as angiogenesis [6–8]. On the other hand, existing PPARα agonists, such as fenofibrate and bezafibrate, sometimes cause adverse effects, especially for use in patients with renal dysfunction or for concomitant use of statin [9–11].

Pemafibrate is a novel selective PPARα modulator (SPPARMα), which has a higher PPARα agonistic activity and selectivity than existing PPARα agonists. Recent evidence indicates that pemafibrate has strong effects on lowering triglycerides and improving atherogenic dyslipidemia without a significant increase in adverse events even in the patients receiving statins [12, 13]. Thus, it is conceivable that pemafibrate can be a useful drug to reduce cardiovascular risk. In this regard, it has been reported that pemafibrate administration improves dyslipidemia and reduces atherosclerotic lesion formation in a mouse model of atherosclerosis [14]. However, little is known about the effect of pemafibrate on the development of ischemic vascular diseases. In the present study, we investigated whether pemafibrate modulates revascularization process in a mouse model of hindlimb ischemia.

Materials and methods

Ethics statement

All animal study protocols were approved by the Institutional Animal Care and Use Committee in Nagoya University.

Materials

Pemafibrate was kindly provided by Kowa Co. Ltd (Nagoya, Japan). Mouse CD31 antibody was purchased from BD Pharmingen (San Jose, CA)(550274). Antibodies of phosphorylated eNOS (Ser-1177)(9571), eNOS (32027) and Tubulin (2144) were purchased from Cell Signaling Technology (Beverly, MA). NG-nitro-l-arginine methyl ester (L-NAME) was purchased from Sigma (St. Louis, MO) (N5751). GW6471 was purchased from Cayman Chemical (11697). Recombinant human FGF21 protein was purchased from R&D system (2539-FG-025). Plasma FGF21 levels were measured by ELISA kit (R & D system)(MF2100) [15]. Plasma adiponectin levels were determined by ELISA kit (Otsuka Pharmaceutical Co. Ltd.)(410713). Adenoviral vectors expressing mouse full-length FGF21 (Ad-FGF21) were constructed under the control of the CMV promoter [16, 17]. Adenoviral vectors expressing β-galactosidase (Ad-βgal) were used as controls [18]. Lipid profiles and plasma glucose were analyzed by enzymatic kits (Wako Pure Chemical Industries, Ltd) (total cholesterol, 439–17501) (triglyceride, 290–63701) (glucose, 439–90901).

Mouse model of hindlimb ischemia

Male wild-type (WT) or eNOS-knockout (eNOS-KO) (Jackson Laboratory) mice at the ages of 6 weeks were fed normal diets containing pemafibrate (0.12 mg/kg/day) or vehicle (Control) for 8 weeks. At the age of 10 weeks, WT or eNOS-KO mice were subjected to unilateral hind limb surgery to remove the left femoral artery and vein under anesthesia [19–22]. In some experiments, Ad-βgal at 1×10⁹ plaque-forming units (pfu) or Ad-FGF21 at 1×10⁹ pfu was intravenously injected into right jugular vein 3 days prior to the surgery as previously described [19, 23]. Hindlimb blood flow was measured by a laser Doppler blood flow analyzer (Moor LDI, Moor Instruments) immediately before surgery and on postoperative days 3, 7, 14 and 28. To avoid data variations caused by ambient light and temperature, hind limb blood flow was expressed as the ratio of left (ischemic) to right (non-ischemic) LDBF. Capillary density within thigh adductor muscle was analyzed by immunohistochemistry [19, 23]. Muscle samples were embedded in OCT compound (Miles, Elkhart, IN) and snap-frozen in liquid nitrogen. Tissue slices (5 μm in thickness) were stained with anti-CD31 antibodies (BD Pharmingen). Fifteen randomly chosen microscopic fields from three different sections in each tissue block were examined for the presence of CD31-positive capillary endothelial cells. Capillary density was expressed as the number of CD31-positive cells per muscle fiber.

Quantification of mRNA levels

Gene expression levels were quantified by real-time PCR method. Total RNA was extracted from skeletal muscle tissues, liver and HUVECs using RNeasy Mini Kit (Qiagen). RNA which had an OD260/280 ratio of 1.8 or greater was used for reverse transcription reaction. cDNA was produced from 0.5 μg total RNA using a Revatra Ace (Toyobo) [24]. PCR was performed with a Bio-Rad real-time PCR detection system using THUNDERBIRD SYBR qPCR Mix as a double-standard DNA-specific dye. Primers were 5'-GCTCCAAGCAGATGCAGCA-3' and 5'-CCGGATGTGAGGCAGCAG-3' for mouse 36B4, 5'-GCTGCTGGAGGACGGTTACA-3' and 5'-CACAGGTCCCCAGGATGTTG-3' for mouse FGF21, 5'-GCCCAGCAACATTATCCAGT-3' and 5'-GGTCAGACTTCCTGCTACGC-3' for mouse LPL, 5'-CGGAGTCCGGGCAGGT-3' and 5'-GCTGGGTAGAGAATGGATGAACA-3' for mouse TNF-α, 5'-GCTACCAAACTGGATATAATCAGGA-3' and 5'-CCAGGTAGCTATGGTACTCCAGAA-3' for mouse IL6, 5'-GCCTGTGTTTTCCTCCTTGC-3' and 5'-CTGCCTAATGTCCCCTTGA-3' for mouse IL1β, 5'-CCACTCACCTGCTGCTACTCAT-3' and 5'-TGGTGATCCTCTTGTAGCTCTCC-3' for mouse MCP1 and 5’-AGGTTGGATGGCAGGC-3’ for mouse adiponectin. All results were normalized to 36B4.

Cell culture

Human umbilical endothelial cells (HUVECs) were cultured in endothelial cell growth medium 2 (Lonza)(EBM-2 (CC-3156), EGM-2 (CC-4176)). HUVECs were cultured in the presence or absence of pemafibrate (10 nM) or recombinant FGF21 protein (10 nM) for the indicated lengths of time.

Assessment of endothelial cell function

The formation of vascular-like structures by HUVECs on growth factor-reduced Matrigel (BD Biosciences) was performed as previously described (20,27). Differentiation was quantified by measuring the area of the “tube-like” networks that form in three randomly chosen fields from each well. Each experiment was repeated three times. Chemotaxis of HUVECs was assessed by transwell assay with polycarbonate membranes coated with fibronectin (Corning)(3415) [25]. HUVECs were added to the upper chamber, and serum-deprived media supplemented with pemafibrate, FGF21 or vehicle was added to the lower chamber. Cells were allowed to migrate through the pores of the membrane for 10 hours. Cell proliferation was assessed by MTS-based assay (Promega)(G3580) [26]. HUVECs were stimulated with pemafibrate, FGF21 or vehicle for the indicated lengths of time under normoxic or hypoxic condition. Hypoxic conditions were generated using an AnaeroPack (5% O₂, 5% CO₂, Mitsubishi GAS Chemical)(3276LJ).

Western blot analysis

Tissue samples were homogenized in lysis buffer containing 1 mM PMSF (Cell Signaling Technology). Immunoblot analysis was performed with antibodies at a 1:1000 dilution, followed by incubation with a secondary antibody conjugated with horseradish peroxidase at a 1:5000 dilution. An ECL Prime Western blotting detection kit (GE healthcare) was used.

Statistical analysis

Data are presented as mean ± S.E. The differences between two groups for variables with normal distributions were evaluated by unpaired Student’s t-test. Differences between three or more groups were evaluated using one-way analysis of variance, with a post-hoc Tukey’s test. A P value < 0.05 denoted the presence of a statistically significant difference. All statistical analyses were performed using SPSS version 18.

Results

Pemafibrate enhances ischemia-induced revascularization in vivo

To examine the effects of pemafibrate on revascularization in response to ischemia, WT mice were fed normal chow diets containing pemafibrate or vehicle (control) followed by subjection to hindlimb ischemia surgery. Fig 1A shows representative laser Doppler blood flow (LDBF) images of hindlimb blood flow before surgery, after surgery, at day 14 and at day 28 after surgery. Pemafibrate administration significantly increased blood flow recovery in ischemic limbs at day 3, 7, 14, 21 or 28 after operation compared with control (Fig 1B).

**A and B**. Effect of pemafibrate administration on blood flow recovery in ischemic limbs of WT mice. Representative Laser Doppler Blood Flow (LDBF) images of limb blood flow in WT mice fed diets containing pemafibrate or vehicle (control) are shown in Fig 1A. Quantitative analysis of the ischemic/non-ischemic LDBF ratio of WT mice receiving control or pemafibrate diet is shown in Fig 1B. *P<0.05. N = 8 in each group. C. Effect of pemafibrate administration on capillary density in ischemic limbs of WT mice. Representative immunostaining of ischemic muscle tissues with anti-CD31 antibody (green) and DAPI (blue) on postoperative day 28. Right panel shows quantitative analyses of capillary density in ischemic muscles of control or pemafibrate-treated WT mice on postoperative day 28. N = 6 in each group. Scale bars show 50 μm. D. Plasma concentration of total cholesterol, triglyceride and glucose of control or pemafibrate-treated WT mice on postoperative day 28. N = 5 in each group (total cholesterol and triglyceride). N = 13 in each group (glucose).

To assess the extent of revascularization at a microcirculatory level, capillary density in non-ischemic or ischemic adductor muscles was assessed by staining with anti-CD31 antibody. Administration of pemafibrate significantly increased the number of CD31-positive cells in ischemic limbs of WT mice (Fig 1C). In contrast, pemafibrate did not affect the number of CD31-positive cells in non-ischemic limbs of WT mice. These findings indicate that pemafibrate promotes ischemia-induced revascularization in vivo. In addition, treatment with pemafibrate significantly reduced plasma triglyceride concentration compared with control, whereas no differences were observed in total cholesterol and glucose levels between two groups (Fig 1D). Treatment with pemafibrate also increased mRNA expression of lipoprotein lipase (LPL), which is a downstream molecule of PPARα, in the liver (S1 Fig).

To test whether pemafibrate directly affects endothelial cell function, HUVECs were placed on a Matrigel matrix and treated with pemafibrate or vehicle. Treatment of HUVECs with pemafibrate increased network areas compared with vehicle (Fig 2A). Treatment with pemafibrate also enhanced migration and proliferation of HUVECs (Fig 2B and 2C). The stimulatory effects of pemafibrate on network formation and migration of HUVECs were canceled by GW6471, which is a specific inhibitor of PPARα (Fig 3B and 3C), indicating that pemafibrate can directly modulate endothelial behavior in a PPARα dependent manner. Treatment of HUVECs with pemafibrate also increased network areas, migration and proliferation compared with vehicle under hypoxic condition (S2 Fig).

Fig 2 — A. Endothelial cell network formation after treatment with pemafibrate. Upper panels show the representative photos of network formation of HUVECs at 16 h after treatment with pemafibate (10 nM) or vehicle. Lower panel shows the quantitative analysis of network area. N = 4 in each group. Scale bars show 1 mm. B. The number of migrated HUVECs at 8 h after treatment with pemafibrate (10 nM) or vehicle. Upper panels show the representative photos of DAPI staining of migrated HUVECs. N = 6 in each group. Scale bars show 200 μm. C. Proliferative activity of HUVECs at 24 h after treatment with pemafibrate (10 nM) or vehicle. N = 8 in each group.

Fig 3 — A. Phosphorylation levels of eNOS in HUVECs after treatment with pemafibrate. Upper panels show the representative blots of phosphorylated eNOS (P-eNOS), eNOS and α-tubulin (Tubulin) at 1 h after treatment with pemafibrate (10 nM) or vehicle. Lower panel shows the quantitative analysis of phosphorylation levels of eNOS relative to Tubulin. N = 4 in each group. **B and C**. Involvement of eNOS and PPARα in pemafibrate-stimulated enhancement of network formation (B) and migration (C) of HUVECs. HUVECs were pretreated with the NOS inhibitor, L-NAME (500 μM) or PPARα specific inhibitor, GW6471 (50 μM), followed by stimulation with pemafibrate (10 nM) or vehicle. N = 8 in each group (B). N = 4 in each group (C). Scale bars show 200 μm. D. Phosphorylation of eNOS in ischemic limb of WT mice fed pemafibrate or control diet. Upper panels show the representative blots of P-eNOS, eNOS and Tubulin at day 28 after surgery. Lower panel shows the quantitative analysis of phosphorylation levels of eNOS relative to Tubulin. N = 7 in each group. E. Effect of pemafibrate administration on blood flow recovery after ischemia in eNOS knockout (eNOS-KO) mice. Quantitative analysis of the ischemic/non-ischemic LDBF ratio of eNOS-KO mice treated with pemafibrate or control diet is shown. N = 10 in each group.

Because eNOS is a key regulator of endothelial cell function [27], we evaluated whether pemafibrate regulates eNOS phosphorylation in HUVECs. Treatment of HUVECs with pemafibrate significantly increased phosphorylation levels of eNOS compared with vehicle culture (Fig 3A). To examine whether pemafibrate enhances endothelial cell function through the eNOS signaling pathway, HUVECs were pretreated with the NOS inhibitor, L-NAME followed by stimulation with pemafibrate or vehicle. Pretreatment with L-NAME abolished pemafibrate-induced enhancement of network formation and migration of HUVECs (Fig 3B and 3C). Consistently, pemafibrate treatment significantly increased phosphorylation levels of eNOS in ischemic adductor muscle, but not in non-ischemic muscle in WT mice compared with control at day 7 and day 28 after surgery (S3 Fig, Fig 3D). Furthermore, pemafibrate treatment had no significant effects on blood flow recovery in eNOS-KO mice throughout the experimental period (Fig 3E). These data suggest that pemafibrate promotes angiogenic responses in vitro and in vivo through the eNOS-dependent mechanism.

Because FGF21 acts as a target gene of PPARα with vasculo-protective effects [28], plasma concentration of FGF21 was measured in control and pemafibrate-treated WT mice. Treatment with pemafibrate robustly increased circulating levels of FGF21 compared with control at day 7 and day 28 after surgery (S4 Fig, Fig 4A). Concomitantly, hepatic expression of FGF21 was significantly higher in pemafibrate-treated WT mice than in control WT mice, whereas no significant difference in FGF21 mRNA levels in ischemic and non-ischemic skeletal muscle was observed between control and pemafibrate-treated mice at day 7 and day 28 after surgery (Fig 4B, S5A and S5B Fig). Pemafibrate treatment did not affect the expression of FGF21 in HUVECs (S5C Fig). In contrast, pemafibrate did not affect circulating levels of the vasculo-protective adipokine adiponectin (S6A Fig). Similarly, pemafibrate had no effects on mRNA expression of adiponectin in epidydimal fat tissue (S6B Fig).

To evaluate whether FGF21 modulates angiogenic response in vivo, adenoviral vectors expressing FGF21 (Ad-FGF21) or control vector (Ad-βgal) were intravenously injected into WT mice 3 days prior to surgery. Systemic administration of Ad-FGF21 significantly increased plasma FGF21 concentration in WT mice compared with Ad-βgal treatment at day 3 and day 17 after adenoviral vector administration (Fig 4C, S7A Fig). Ad-FGF21 administration also increased the expression of FGF21 in ischemic skeletal muscle of WT mice compared with Ad-βgal treatment at day 3 after adenoviral vector injection (S7B Fig).

Ad-FGF21 administration significantly enhanced blood flow recovery in ischemic limbs of WT mice compared with Ad-βgal treatment (Fig 4D). The number of CD31-positive cells was significantly higher in Ad-FGF21-treated mice compared with Ad-βgal-treated mice (Fig 4E). Furthermore, treatment with FGF21 protein promoted network formation, migration and proliferation of HUVECs compared with vehicle (Fig 5A, 5B and 5C). These results indicate that increased levels of FGF21 by pemafibrate treatment could contribute to an increased blood flow recovery of WT mice.

Fig 5 — A. Quantitative analysis of network area at 16 h after treatment with FGF21 protein (10 nM) or vehicle. Upper panels show representative photos of network formation of HUVECs. N = 5 in each group. Scale bars show 1 mm. B. The number of migrated HUVECs at 8 h after treatment with FGF21 (10 nM) or vehicle. Upper panels show representative photos of DAPI staining of migrated HUVECs. N = 6 in each group. Scale bars show 100 μm. C. Proliferative activity of HUVECs at 24 h after treatment with FGF21 (10 nM) or vehicle. N = 10 in each group.

Finally, we evaluated whether FGF21 modulates eNOS phosphorylation in vitro and in vivo. Treatment with FGF21 protein increased eNOS phosphorylation in HUVECs compared with control (Fig 6A). NOS inhibition by pretreatment with L-NAME blocked the stimulatory effects of FGF21 on endothelial cell differentiation, migration and proliferation (Fig 6B, 6C and 6D). Ad-FGF21 treatment significantly increased phosphorylation levels of eNOS in ischemic muscle, but not in non-ischemic muscle in WT mice compared with Ad-βgal treatment (Fig 6E). Furthermore, Ad-FGF21 did not affect blood flow recovery after surgery in eNOS-KO mice (Fig 6F). These data indicate that FGF21 promotes endothelial cell function and ischemia-induced revascularization through the eNOS-dependent mechanism.

Fig 6 — A. Phosphorylation of eNOS in HUVECs after treatment with FGF21. Upper panels show the representative blots of phosphorylated eNOS (P-eNOS), eNOS and α-tubulin (Tubulin) at 1 h after treatment with FGF21 (10 nM) or vehicle. Lower figure shows the quantitative analysis of phosphorylation levels of eNOS relative to Tubulin. N = 5 in each group. **B-D**. Involvement of eNOS in FGF21-induced enhancement of network structure (B), migration (C) and proliferation (D) of HUVECs. HUVECs were pretreated with the NOS inhibitor, L-NAME (500 μM) followed by stimulation with FGF21 (10 nM) or vehicle. N = 8 in each group (B and C), N = 10 in each group (D). Scale bars show 1 mm (B) and 200 μm (C). E. Phosphorylation of eNOS in non-ischemic and ischemic limb of WT mice treated with Ad-FGF21 or Ad-βgal. Upper panels show the representative blots of P-eNOS, eNOS and Tubulin at day 14 after surgery. N = 4 in each group. F. Effect of FGF21 on blood flow recovery after ischemia in eNOS knockout (eNOS-KO) mice. Quantitative analysis of the ischemic/non-ischemic LDBF ratio of eNOS-KO mice treated with Ad-FGF21 or Ad-βgal is shown. N = 5 in each group.

Discussion

This study provides the first evidence that a novel selective PPARα agonist, pemafibrate promotes endothelial cell function and revascularization under conditions of ischemia. Systemic administration of pemafibrate enhanced blood flow recovery and capillary density in ischemic limbs of WT mice. Treatment with pemafibrate stimulated network formation, migration and proliferative activity of cultured endothelial cells under conditions of normoxia or hypoxia. The stimulatory effects of pemafibrate on endothelial cell function were abolished by PPARα inhibition, indicating that pemafibrate can modulate endothelial behavior in a PPARα dependent manner. Importantly, although FGF21 is a target gene of PPARα, pemafibrate did not affect FGF21 expression in cultured endothelial cells. Thus, it is likely that pemafibrate can affect endothelial cell function in a FGF21 independent manner. These data suggest that pemafibrate can directly modulate endothelial behavior via a PPARα signaling mechanism that is independent of FGF21 induction. Pemafibrate administration also led to significant increases in hepatic expression level of FGF21 and plasma level of FGF21. Systemic administration of FGF21 enhanced ischemia-induced revascularization in WT mice. Treatment of endothelial cells with FGF21 promoted network formation, migratory activity and growth. Thus, it is likely that pemafibrate stimulates revascularization process after ischemia through at least two mechanisms: direct modulation of endothelial cell behavior and enhancement of pro-angiogenic factor FGF21-mediated endothelial cell function.

FGF21 is an endocrine factor that is expressed in several tissues including liver and skeletal muscle [29]. In the present study, treatment with pemafibrate robustly increased FGF21 expression in the liver and plasma levels of FGF21. In contrast, pemafibrate had no effects on FGF21 expression in skeletal muscle tissue, consistent with a previous report [30]. It has been shown that liver is the major source of circulating FGF21 [31]. Thus, these data suggest that pemafibrate administration contributes to elevation of circulating levels of liver-derived FGF21, which affects the function of endothelium in an endocrine manner. However, Thus, future studies using FGF21 deficient mice or FGF21 inhibitors will be required to clarify whether FGF21 is essential for the pro-angiogenic effects of pemafibrate.

It is well known that eNOS plays a pivotal role in regulation of endothelial function and angiogenic response under normal physiological and ischemic conditions [32–34]. Our data showed that pemafibrate promoted endothelial cell network formation and migration in an eNOS-dependent manner. This is consistent with the previous reports showing that fenofibrate activates eNOS in cultured endothelial cells [35, 36]. Our data also showed that FGF21 enhanced endothelial cell function through the eNOS signaling pathway. Furthermore, our data showed that systemic delivery of pemafibrate or FGF21 enhanced eNOS phosphorylation in ischemic skeletal muscle tissues, but not in non-ischemic skeletal muscles. These findings are in agreement with the results that pemafibrate or FGF21 is effective at increasing capillary density only in ischemic tissue but not non-ischemic tissue. However, our in vitro data showed that pemafibrate promoted endothelial cell function under both normoxic and hypoxic conditions. The reason for the discrepancy between in vivo and in vitro effects of pemafibrate on angiogenic response is unknown, and this requires future investigation. Of note, the stimulatory effect of pemafibrate or FGF21 on blood flow recovery in ischemic limbs was abolished under conditions of eNOS deficiency. In addition, pemafibrate dramatically increased circulating levels of FGF21 in mice. Collectively, our data suggest that pemafibrate can promote revascularization in response to ischemia, at least in part, by its ability to promote endothelial cell function through direct and FGF21-mediated activation of eNOS in endothelial cells (S8 Fig).

We previously reported that fenofibrate promotes revascularization in response to ischemia in mice through upregulation of the vasculo-protective adipokine adiponectin [37]. In contrast, the present data demonstrated that pemafibrate had no effects on plasma levels of adiponectin in WT mice. It has also been shown that FGF21 increases adiponectin production in adipose tissue, thereby leading to enhanced levels of circulating adiponectin [38, 39]. Our data showed that pemafibrate did not affect the expression of adiponectin in adipose tissue of mice despite a dramatic increase in circulating levels of FGF21. These findings are consistent with a clinical report showing that pemafibrate increases plasma levels of FGF21 without affecting circulating adiponectin levels [40]. Thus, it is conceivable that the salutary effect of pemafibrate on angiogenic response in vivo is independent of adiponectin.

FGF21 acts as a multifunctional regulator of metabolism and cardiovascular function. FGF21 is reported to improve insulin sensitivity and hypertriglyceridemia [38, 39, 41]. It has also been reported that FGF21 protects against atherosclerosis in a mouse model of atherosclerosis [28, 42]. We have previously reported that FGF21 attenuates adverse cardiac remodeling in mice after myocardial infarction [42]. The present data indicate that FGF21 promotes angiogenic response to ischemic injury. Thus, these data propose that pemafibrate may exert beneficial actions on lipid and glucose metabolism, and cardiovascular disorders partly via upregulation of FGF21 expression.

In conclusion, our present study provides the first evidence that a newly developed SPPARMα, pemafibrate promotes pro-angiogenic response by modulating endothelial cell function. Thus, pemafibrate could be a potentially beneficial drug for prevention or treatment of peripheral arterial disease.

Supporting information

S1 Fig. Pemafibrate increases mRNA levels of PPARα target gene, lipoprotein lipase (LPL) in the liver.

N = 3 in each group.

(PDF)

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S2 Fig. Pemafibrate promotes endothelial cell function under hypoxic condition.

A. Endothelial cell network formation after treatment with pemafibrate under hypoxic condition. Upper panels show the representative photos of network formation of HUVECs at 8 h after treatment with pemafibate (10 nM) or vehicle. Lower panel shows the quantitative analysis of network area. N = 8 in each group. Scale bars show 1 mm. B. The number of migrated HUVECs at 8 h after treatment with pemafibrate (10 nM) or vehicle under hypoxic condition. Upper panels show the representative photos of DAPI staining of migrated HUVECs. N = 6 in each group. Scale bars show 200 μm. C. Proliferative activity of HUVECs at 8 h after treatment with pemafibrate (10 nM) or vehicle. N = 10 in each group.

(PDF)

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S3 Fig. Phosphorylation of eNOS in non-ischemic and ischemic limb of WT mice fed pemafibrate or control diet.

Upper panels show the representative blots of P-eNOS, eNOS and Tubulin at day 7 after surgery. Lower panel shows the quantitative analysis of phosphorylation levels of eNOS relative to eNOS. N = 4 in each group.

(PDF)

Click here for additional data file.^{(28.2KB, pdf)}

S4 Fig. Plasma concentration of FGF21 of control or pemafibrate-treated WT mice at day 7 after operation as evaluated by ELISA system.

N = 5 in each group.

(PDF)

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S5 Fig. Effects of pemafibrate on FGF21 expression in skeletal muscle and endothelial cells.

A and B. Pemafibrate did not affect the expression of FGF21 in non-ischemic and ischemic skeletal muscle at day 7 (A) and day 28 (B) after surgery. N = 8 in each group (A). N = 5 in each group (B). C. Treatment of HUVECs with pemafibrate had no effects on FGF21 expression. N = 5 in each group.

(PDF)

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S6 Fig. Effect of pemafibrate on plasma adiponectin (APN) and adipose tissue APN mRNA levels.

A. Plasma concentration of APN in WT mice fed control or pemafibrate diet. B. The mRNA expression of APN in epididymal fat tissue of WT mice fed control or pemafibrate diet. N = 8 in each group.

(PDF)

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S7 Fig. FGF21 levels in plasma and skeletal muscle after treatment with Ad-FGF21 or Ad-βgal.

A. Plasma concentration of FGF21 in WT mice at day 17 after Ad-FGF21 or Ad-βgal administration as evaluated by ELISA system. N = 8 in each group. B. The mRNA levels of FGF21 in ischemic skeletal muscle at day 3 (N = 5 in each group) and day 17 (N = 8 in each group) after Ad-FGF21 or Ad-βgal administration.

(PDF)

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S8 Fig. Proposed scheme of the possible mechanisms by which pemafibrate modulates endothelial cell function and revascularization process.

Pemafibrate directly activates eNOS signaling pathway in endothelium. Pemafibrate treatment leads to increases in hepatic FGF21 expression and circulating FGF21 levels, which in turn promote eNOS activation in endothelium. These two pathways are involved in regulation of endothelial cell function and revascularization.

(PDF)

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S1 Raw Images

(PDF)

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Acknowledgments

We would like to thank Yoko Inoue and Minako Tatsumi for technical assistance.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by Grant-in-Aid for Scientific Research (2620H00571 to N.O., 2617H04175 to N.O., 2616K09512 to K.O.); grants from Takeda Science Foundation (2600007594 and 2600007051 to N.O); and a grant from the Suzuken Memorial Foundation (2600007887 to N.O.).

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PLoS One. doi: 10.1371/journal.pone.0235362.r001

Decision Letter 0

Masuko Ushio-Fukai

18 Mar 2020

PONE-D-20-03337

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

PLOS ONE

Dear Dr. Koji Ohashi

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Reviewer #1: The manuscript, a title “a novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanism” by Kawanishi H. et al is very interesting but need to present additional supporting data to prove the hypothesis.

General comments.

The main idea is that pemafibrate increase liver FGF21 and result in increasing circulated-FGF21 in blood which induces phosphorylation of eNOS to enhance ischemia-induced revascularization.

However, authors did not discuss why pemafibrate is effective only in ischemic tissue but not non-ischemic tissue. In addition, it is not clear even though authors suggested at least two mechanisms of pemafibrate at discussion. Please clarify how pemafibrate directly modulates endothelial behavior because authors only presented second effects of pemafibrate via FGF21 in current manuscript.

Authors presented in vitro effects of pemafibrate using HUVECs and showed that pemafibrate increases angiogenic characteristics such as cell migration, network-like tube formation, and proliferation. In vivo experiment, authors showed that pemafibrate was effective only in ischemic-hind limb tissue. It is not consistent between in vitro experiments and in vivo experiments because authors did all in vitro experiments under nomoxic condition.

In addition, authors used 28 days ischemic-limb tissues to show p-eNOS but 1h samples in HUVECs. It is highly possible that FGF21 increase p-eNOS early time point and then the p-eNOS plays a role to enhance revascularization pathway. It might be already finished revascularization at end point 28 days dependent on laser doppler images. Please show all time course for p-eNOS using hind limb ischemic tissues because it is important to check p-eNOS kinetics to enhance revascularization.

Please show ad-FGF21 expression levels in skeletal muscle tissues with time course.

Need to have more detail method section. For example, how authors measured plasma concentration of total choresterol, triglycerol, and glucose.

Please show p-eNOS levels in non-ischemic limb tissues with ischemic tissues in parallel.

Please clarify when authors measured plasma FGF21 concentration after administrating ad-FGF21.

The quantification of network area in Figure 2A is not matched with images. Please change with representative images.

Please show all images for tube formation and migration assays.

Please normalize p-eNOS levels by total eNOS levels instead of tubulin to clarify whether eNOS expression does not change by pemafibrate or FGF21.

Please show FGF21 levels in HUVEC treated with or without pemafibrate.

Please show plasma FGF21 levels with time course in non-ischemic and ischemic tissue.

Please clarify whether FGF21 is an initial key messenger of pemafibrate by showing rescue effect with FGF21 inhibitor or antibody.

Please show effects of pemafibrate in vitro under hypoxic condition to verify in vivo hind limb results.

The conclusion is overestimated because authors did not show any data for lipid metabolism.

Reviewer #2: The manuscript entitled "A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms" is well written. However, the authors need to clarify the following concerns.

1. Throughout the manuscript the authors have referred the figure 6 as figure 5 which is confusing and need serious attention.

2. The IF staining of CD31 in muscle is not much informative. It is very difficult to understand ischemic region without nuclear staining. IHC staining of the same will be more acceptable.

3. The quality of blots are not good enough, such as in figure 3D, 6E.

4. The bar graph for eNOS phosphorylation should be expressed in term of p-eNOS/total eNOS.

5. According to authors, pemafibrate stimulates revascularization through direct effect on endothelial cells. But no mechanism has been provided or predicted. Pemafibrate stimulated revascularization through increased expression of FGF21 does not explain the in-vitro effect.

6. The effect of pemafibrate on FGF21 is well known and the effect of FGF21 on endothelial cell proliferation through eNOS has also been reported. Therefore, the significance of the manuscript can be enhanced if the authors could find the mechanism of direct effect of pemafibrate on ECs.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Jun 25;15(6):e0235362. doi: 10.1371/journal.pone.0235362.r002

Author response to Decision Letter 0

1 May 2020

Reviewer #1:

The manuscript, a title “a novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanism” by Kawanishi H. et al is very interesting but need to present additional supporting data to prove the hypothesis.

Response: We thank the reviewer for these positive comments.

General comments.

The main idea is that pemafibrate increase liver FGF21 and result in increasing circulated-FGF21 in blood which induces phosphorylation of eNOS to enhance ischemia-induced revascularization. However, authors did not discuss why pemafibrate is effective only in ischemic tissue but not non-ischemic tissue. In addition, it is not clear even though authors suggested at least two mechanisms of pemafibrate at discussion. Please clarify how pemafibrate directly modulates endothelial behavior because authors only presented second effects of pemafibrate via FGF21 in current manuscript.

Response: We thank the reviewer for these suggestions. We examined the effects of pemafibrate on HUVEC behaviors in vitro under hypoxic condition. Network formation, migration and proliferation of HUVECs were promoted by pemafibrate treatment even under hypoxic condition. Thus, it is conceivable that pemafibrate treatment can enhance the revascularization and capillary density in ischemic limb of mice. These data are included in Supplemental Figure 2 and Results section (Page 9, line 16-18) in the revised manuscript.

We found that systemic delivery of pemafibrate or FGF21 enhanced eNOS phosphorylation in ischemic skeletal muscle tissues, but not in non-ischemic skeletal muscles. These findings are in agreement with the results that pemafibrate or FGF21 is effective at increasing capillary density only in ischemic tissue but not non-ischemic tissue. These data are included in Figure 3D, Supplemental Figure 3 , Results section (Page 9, line 26 – Page 10, line 3) and Discussion section (Page 13, line 2-6) in the revised manuscript.

In addition, we found that the stimulatory effects of pemafibrate on network formation and migration of HUVECs were abolished by GW6471, which is a specific inhibitor of PPARα, indicating that pemafibrate can directly modulate endothelial behavior in a PPARα dependent manner. These data are included in Figure 3B, 3C, Result section (Page 9, line 13-18) and Discussion section (Page 11, line 26 – Page 12, line 3) in the revised manuscript.

Response: We thank the reviewer for these important suggestions. We examined the effects of pemafibrate on HUVEC behaviors in vitro under hypoxic condition. Network formation, migration and proliferation of HUVECs were promoted by pemafibrate treatment even under hypoxic condition. Thus, it is conceivable that pemafibrate treatment can enhance the revascularization and capillary density in ischemic limb of mice. These data are included in Supplemental Figure 2 and Results section (Page 9, line 16-18) in the revised manuscript.

Response: As the reviewer pointed out, p-eNOS could increase at earlier time point. Thus, we checked p-eNOS in skeletal muscles at day 7 after surgery. The levels of p-eNOS were higher in ischemic limb of pemafibrate-treated mice than in that of control mice at day 7. This result is now shown in Supplemental Figure 3 and Result section (Page 9, line 26 – Page 10, line 3) in the revised manuscript.

Please show ad-FGF21 expression levels in skeletal muscle tissues with time course.

Response: We thank the reviewer for the useful suggestion. We measured FGF21 expression of skeletal muscles both at day 3 and day 17 after adenoviral vector injection. The mRNA levels of FGF21 increased at skeletal muscle at 3 days after Ad-FGF21 injection compared with control Ad-βgal injection. The mRNA levels of FGF21 also tended to be higher at skeletal muscle at 17 days after Ad-FGF21 injection than Ad-βgal injection, but it was not statistically significant. These results are now included in Supplemental Figure 7B and Result section (Page 10, line 25 – Page 11, line 1).

Need to have more detail method section. For example, how authors measured plasma concentration of total choresterol, triglycerol, and glucose.

Response: We thank the reviewer for these suggestions. We prodived the more detailed description for measurement of total cholesterol, triglyceride and glucose in Materials and Methods sections (Page 5, line 15-16) in the revised manuscript.

Please show p-eNOS levels in non-ischemic limb tissues with ischemic tissues in parallel.

Response: According to the reviewer’s suggestion, we evaluated the p-eNOS levels in non-ischemic limb tissues, too. These results are now shown in Figure 3D and Supplemental Figure 3 in the revised manuscript.

Please clarify when authors measured plasma FGF21 concentration after administrating ad-FGF21.

Response: We measured plasma FGF21 concentration at day 3 after Ad-FGF21 administration. In addition, we evaluated plasma FGF21 concentration at day 17 after Ad-FGF21 administration. These data and points are included in Figure 4C, Supplemental Figure 7A and Result section (Page 10, line 22-25) in the revised manuscript.

The quantification of network area in Figure 2A is not matched with images. Please change with representative images.

Response: According to the reviewer’s suggestion, we added new representative photos of network formation in Figure 2A.

Please show all images for tube formation and migration assays.

Response: According to the reviewer’s suggestions, we added all images for tube formation and migration assay in the revised manuscript.

Please normalize p-eNOS levels by total eNOS levels instead of tubulin to clarify whether eNOS expression does not change by pemafibrate or FGF21.

Response: According to the reviewer’s suggestion, we normalized p-eNOS levels by total eNOS levels in Figure 3A, 3D, 6A, 6E and Supplemental Figure 3 in the revised manuscript.

Please show FGF21 levels in HUVEC treated with or without pemafibrate.

Response: We thank the reviewer for an important suggestion. We checked mRNA levels of FGF21 in the presence or absence of pemafibrate in HUVECs. Treatment with pemafibrate had no effect on the expression levels of FGF21 in HUVECs. These findings are now shown in Supplemental Figure 5C and Result section in our revised manuscript (Page 10, line 15-16).

Please show plasma FGF21 levels with time course in non-ischemic and ischemic tissue.

Response: We thank the reviewer for the suggestion. We measured plasma FGF21 levels at day 7 and day 28 after surgery. Treatment with pemafibrate increased plasma FGF21 levels both at day 7 and day 28 compared with control. These results are shown Supplemental Figure 4, Figure 4A and Result section in a revised manuscript (Page 10, line 9-11). In contrast, there were no difference in the mRNA levels of FGF21 in non-ischemic and ischemic skeletal muscle at day 7 and day 28 after surgery. These data are now shown in Supplemental Figure 5A, 5B and Result section in a revised manuscript (Page 10, line 11-15).

Please clarify whether FGF21 is an initial key messenger of pemafibrate by showing rescue effect with FGF21 inhibitor or antibody.

Response: It is important to clarify whether FGF21 is a key regulator of pemafibrate in angiogenic response in vivo. Thus, we should investigate the contribution of FGF21 to pemafibrate-stimulated revascularization by using FGF21 deficient mice or FGF21 inhibitors. The time limit to revise our manuscript was May 2, which was around 6 weeks later after the first decision. It takes more than 6 weeks to perform these experiments. In addition, we believe that this is beyond the scope of this paper. Thus, future studies using FGF21 deficient mice or FGF21 inhibitors will be required to clarify whether FGF21 is essential for the pro-angiogenic effects of pemafibrate. These points are included in the discussion section in the revised manuscript (Page 12, line 19-21).

Please show effects of pemafibrate in vitro under hypoxic condition to verify in vivo hind limb results.

Response: We thank the reviewer for these suggestions. We examined the effects of pemafibrate on HUVEC behaviors in vitro under hypoxic condition. Network formation, migration and proliferation of HUVECs were promoted by pemafibrate treatment even under hypoxic condition. These findings are now included in Supplemental Figure 2, Result section (Page 9, line 16-18) and Methods section (Page 7, line 22-25) in the revised manuscript.

The conclusion is overestimated because authors did not show any data for lipid metabolism.

Response: We agree with the reviewer. We deleted the sentence “Clinically, pemafibrate treatment can ameliorate the atherogenic lipid profiles.” We also changed from the phrase “cardiovascular disease” to “peripheral arterial disease” in Discussion section (Page 14, line 9-10). In addition, we deleted the phrase “among patients with dyslipidemia” in conclusion in the abstract.

Reviewer #2:

The manuscript entitled "A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms" is well written. However, the authors need to clarify the following concerns.

1. Throughout the manuscript the authors have referred the figure 6 as figure 5 which is confusing and need serious attention.

Response: We thank the reviewer for carefully reading our manuscript. We corrected our mistake in the revised manuscript.

2. The IF staining of CD31 in muscle is not much informative. It is very difficult to understand ischemic region without nuclear staining. IHC staining of the same will be more acceptable.

Response: We thank the reviewer for an important suggestion. We added photos of nuclear staining (DAPI) with CD31 staining in Figure 1C and 4E in the revised manuscript.

3. The quality of blots are not good enough, such as in figure 3D, 6E.

Response: We thank the reviewer for these suggestions. The quality of blots has been improved in Figure 3D and 6E in the revised manuscript.

4. The bar graph for eNOS phosphorylation should be expressed in term of p-eNOS/total eNOS.

Response: According to the reviewer’s suggestion, we evaluated p-eNOS levels in p-eNOS/total eNOS in Figure 3A, 3D, 6A, 6E and Supplemental Figure 3 in the revised manuscript.

Response: We thank the reviewer for these important suggestions. we found that the stimulatory effects of pemafibrate on network formation and migration of HUVECs were abolished by GW6471, which is a specific inhibitor of PPARα, indicating that pemafibrate can directly modulate endothelial behavior in a PPARα dependent manner. These data are included in Figure 3B, 3C and Result section (Page 9, line 13-16) in the revised manuscript.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0235362.r003

Decision Letter 1

Masuko Ushio-Fukai

28 May 2020

PONE-D-20-03337R1

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

PLOS ONE

Dear Dr. Koji Ohashi

Furthermore, please provide the blots or gels include original uncropped blot/gel image data as a supplement file. The blots or gels provided by the authors in the previous review have been already cropped, which are not considered as original ones.

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Masuko Ushio-Fukai, PhD

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Authors addressed almost reviewer’s comments and all responses are reasonable.

However, reviewer would like to request minor revision as following.

1. It is very important to reproduce authors’ research. Therefore, please provide exact information (company and catalog number, if you modified procedures, please describe it) of experimental assay kits.

2. In the same line, please clarify hypoxic condition (oxygen concentration) and provide a positive control to show that experimental system works well.

3. All images should have scale bars.

4. Almost images are very small. Please provide enlarged images with scale bars.

5. Western blotting images also should have molecular markers.

6. Please carefully check authors’ response. Because authors have wrong response about a part of first comment. Reviewer guess authors just copied and pasted it from response about second comment.

Reviewer #2: Although the authors have tried to answer the queries raised in previous review, still some questions have remain unanswered or not satisfactory.

1. CD31 staining in ischemic and non ischemic muscle is still not convincing. There are no bar graphs in the figures.

2. Since, pemafibrate stimulated EC function in both normoxic and hypoxic condition in vitro, it fails to explain the effect of pemafibrate on ischemic tissue only.

3. Treatment of EC with pemafibrate and GW6471 together could not explain the direct effect of pemafibrate on EC function independent of FGF21 since FGF21 is downstream to PPARα.

**********

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Reviewer #1: No

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

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PLoS One. 2020 Jun 25;15(6):e0235362. doi: 10.1371/journal.pone.0235362.r004

Author response to Decision Letter 1

2 Jun 2020

Response to Reviewer 1

Authors addressed almost reviewer’s comments and all responses are reasonable.

However, reviewer would like to request minor revision as following.

Response: We thank the reviewer for the positive comment.

Response: We thank the reviewer for the important suggestion. We provided vendor names, catalog numbers of experimental assay kits in our revised manuscript.

2. In the same line, please clarify hypoxic condition (oxygen concentration) and provide a positive control to show that experimental system works well.

Response: We thank the reviewer for the important suggestion. HUVECs were exposed to 5% oxygen concentration by using Anaeropack system. We previously exposed cultured cardiomyocytes (rat neonatal ventricular myocytes) and HUVECs to hypoxia using this system (Ref1-5). Cardiomyocytes led to more than 20% apoptosis after 12 h hypoxia following 24 h normoxia stimulation in this system. We described the information of oxygen concentration and carbon dioxide concentration in the revised manuscript (Page 8, line 4).

3. All images should have scale bars.

Response: We thank the reviewer for the important suggestion. All photos have scale bars in our revised Figure and Supplemental Figure.

4. Almost images are very small. Please provide enlarged images with scale bars.

Response: We thank the reviewer for the important suggestion. We enlarged photos in the revised Figures.

5. Western blotting images also should have molecular markers.

Response: We thank the reviewer for the important suggestion. We showed molecular weight markers of Western blots in our revised Figures and Supplemental Figures.

6. Please carefully check authors’ response. Because authors have wrong response about a part of first comment. Reviewer guess authors just copied and pasted it from response about second comment.

Response: We regret our wrong response in first comment. The reviewer suggested discussion of the reason why pemafibrate promotes angiogenic response only under ischemic condition. Thus, we discussed this point in Discussion section (Page 13, line 14 -17).

References

1. Ogura Y, Ouchi N, Ohashi K, Shibata R, Kataoka Y, Kambara T, Kito T, Maruyama S, Yuasa D, Matsuo K, Enomoto T, Uemura Y, Miyabe M, Ishii M, Yamamoto T, Shimizu Y, Walsh K, Murohara T. Therapeutic impact of follistatin-like 1 on myocardial ischemic injury in preclinical animal models. Circulation. 2012;126:1728-1738

2. Kataoka Y, Shibata R, Ohashi K, Kambara T, Enomoto T, Uemura Y, Ogura Y, Yuasa D, Matsuo K, Nagata T, Oba T, Yasukawa H, Numaguchi Y, Sone T, Murohara T, Ouchi N. Omentin prevents myocardial ischemic injury through amp-activated protein kinase- and akt-dependent mechanisms. J Am Coll Cardiol. 2014;63:2722-2733

3. Yuasa D, Ohashi K, Shibata R, Mizutani N, Kataoka Y, Kambara T, Uemura Y, Matsuo K, Kanemura N, Hayakawa S, Hiramatsu-Ito M, Ito M, Ogawa H, Murate T, Murohara T, Ouchi N. C1q/tnf-related protein-1 functions to protect against acute ischemic injury in the heart. FASEB J. 2016;30:1065-1075

4. Ohashi K, Enomoto T, Joki Y, Shibata R, Ogura Y, Kataoka Y, Shimizu Y, Kambara T, Uemura Y, Yuasa D, Matsuo K, Hayakawa S, Hiramatsu-Ito M, Murohara T, Ouchi N. Neuron-derived neurotrophic factor functions as a novel modulator that enhances endothelial cell function and revascularization processes. J Biol Chem. 2014;289:14132-14144

5. Joki Y, Ohashi K, Yuasa D, Shibata R, Kataoka Y, Kambara T, Uemura Y, Matsuo K, Hayakawa S, Hiramatsu-Ito M, Kanemura N, Ito M, Ogawa H, Daida H, Murohara T, Ouchi N. Neuron-derived neurotrophic factor ameliorates adverse cardiac remodeling after experimental myocardial infarction. Circ Heart Fail. 2015;8:342-351

Response to Reviewer #2

Although the authors have tried to answer the queries raised in previous review, still some questions have remain unanswered or not satisfactory.

Response:

1. CD31 staining in ischemic and non ischemic muscle is still not convincing. There are no bar graphs in the figures.

Response: We thank the reviewer for the important comment. We showed the quantitative graphs on the right side of representative CD31 staining.

2. Since, pemafibrate stimulated EC function in both normoxic and hypoxic condition in vitro, it fails to explain the effect of pemafibrate on ischemic tissue only.

Response: The reviewer raised a good point. The reason for the discrepancy between in vivo and in vitro effects of pemafibrate on angiogenic response is unknown. This requires future investigation. These points are included in the discussion section in the revised manuscript (Page 13. Line 14-17).

3. Treatment of EC with pemafibrate and GW6471 together could not explain the direct effect of pemafibrate on EC function independent of FGF21 since FGF21 is downstream to PPARα.

Response: We thank the reviewer for these important suggestions. The stimulatory effects of pemafibrate on endothelial cell function were abolished by PPARα inhibition, indicating that pemafibrate can modulate endothelial behavior in a PPARα dependent manner. Importantly, although FGF21 is a target gene of PPARα, pemafibrate did not affect FGF21 expression in cultured endothelial cells. Thus, it is likely that pemafibrate can affect endothelial cell function in a FGF21 independent manner. These data suggest that pemafibrate can directly modulate endothelial behavior via a PPARα signaling mechanism that is independent of FGF21 induction. These points are included in the discussion section in the revised manuscript (Page 12, line 5 - 12).

Attachment

Submitted filename: Response to Reviewers 060220.doc

Click here for additional data file.^{(50KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0235362.r005

Decision Letter 2

Masuko Ushio-Fukai

3 Jun 2020

PONE-D-20-03337R2

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

PLOS ONE

Dear Dr. Dr Koji Ohashi

Thank you for submitting your manuscript to PLOS ONE. Although revised manuscript scientifically improved and authors responded to each reviewer's concern satisfactory, supplemental data unfortunately still did not include the "Original and Uncropped and Unadjusted blot/gel image data". Please provide the original gels before cutting and cropping for each figure. For example, there were space or line between blots in Supplement. Please provide the gels/blots without space or lines.

(Instruction from PLOS ONE)

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Kind regards,

Masuko Ushio-Fukai, PhD

Academic Editor

PLOS ONE

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Jun 25;15(6):e0235362. doi: 10.1371/journal.pone.0235362.r006

Author response to Decision Letter 2

9 Jun 2020

Academic editor mentioned like below. According to academic editor's comments, we changed only Raw Images of gel data.

"Thank you for submitting your manuscript to PLOS ONE. Although revised manuscript scientifically improved and authors responded to each reviewer's concern satisfactory, supplemental data unfortunately still did not include the "Original and Uncropped and Unadjusted blot/gel image data". Please provide the original gels before cutting and cropping for each figure. For example, there were space or line between blots in Supplement. Please provide the gels/blots without space or lines."

PLoS One. doi: 10.1371/journal.pone.0235362.r007

Decision Letter 3

Masuko Ushio-Fukai

15 Jun 2020

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

PONE-D-20-03337R3

Dear Dr. Koji Ohashi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Masuko Ushio-Fukai, PhD

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0235362.r008

Acceptance letter

Masuko Ushio-Fukai

17 Jun 2020

PONE-D-20-03337R3

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

Dear Dr. Ohashi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Pemafibrate increases mRNA levels of PPARα target gene, lipoprotein lipase (LPL) in the liver.

N = 3 in each group.

(PDF)

Click here for additional data file.^{(7.6KB, pdf)}

S2 Fig. Pemafibrate promotes endothelial cell function under hypoxic condition.

(PDF)

Click here for additional data file.^{(29.8KB, pdf)}

S3 Fig. Phosphorylation of eNOS in non-ischemic and ischemic limb of WT mice fed pemafibrate or control diet.

(PDF)

Click here for additional data file.^{(28.2KB, pdf)}

S4 Fig. Plasma concentration of FGF21 of control or pemafibrate-treated WT mice at day 7 after operation as evaluated by ELISA system.

N = 5 in each group.

(PDF)

Click here for additional data file.^{(7.5KB, pdf)}

S5 Fig. Effects of pemafibrate on FGF21 expression in skeletal muscle and endothelial cells.

(PDF)

Click here for additional data file.^{(41.6KB, pdf)}

S6 Fig. Effect of pemafibrate on plasma adiponectin (APN) and adipose tissue APN mRNA levels.

A. Plasma concentration of APN in WT mice fed control or pemafibrate diet. B. The mRNA expression of APN in epididymal fat tissue of WT mice fed control or pemafibrate diet. N = 8 in each group.

(PDF)

Click here for additional data file.^{(29.6KB, pdf)}

S7 Fig. FGF21 levels in plasma and skeletal muscle after treatment with Ad-FGF21 or Ad-βgal.

(PDF)

Click here for additional data file.^{(31.4KB, pdf)}

S8 Fig. Proposed scheme of the possible mechanisms by which pemafibrate modulates endothelial cell function and revascularization process.

(PDF)

Click here for additional data file.^{(8.8KB, pdf)}

S1 Raw Images

(PDF)

Click here for additional data file.^{(4.2MB, pdf)}

Attachment

Submitted filename: PONE-D-20-03337-review.docx

Click here for additional data file.^{(20.2KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

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Attachment

Submitted filename: PONE-D-20-03337-R1 review.docx

Click here for additional data file.^{(13.4KB, docx)}

Attachment

Submitted filename: Response to Reviewers 060220.doc

Click here for additional data file.^{(50KB, doc)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

A novel selective PPARα modulator, pemafibrate promotes ischemia-induced revascularization through the eNOS-dependent mechanisms

Hiroshi Kawanishi

Koji Ohashi

Hayato Ogawa

Naoya Otaka

Tomonobu Takikawa

Lixin Fang

Yuta Ozaki

Mikito Takefuji

Toyoaki Murohara

Noriyuki Ouchi

Roles

Abstract

Objective

Methods and results

Conclusion

Introduction

Materials and methods

Ethics statement

Materials

Mouse model of hindlimb ischemia

Quantification of mRNA levels

Cell culture

Assessment of endothelial cell function

Western blot analysis

Statistical analysis

Results

Pemafibrate enhances ischemia-induced revascularization in vivo

Fig 1. Pemafibrate promotes blood flow recovery and capillary density in ischemic limbs of WT mice.

Fig 2. Pemafibrate promotes endothelial cell function in vitro.

Fig 3. Pemafibrate promotes angiogenic response through the eNOS-dependent pathway.

Fig 4. FGF21 stimulates revascularization in ischemic limbs of WT mice.

Fig 5. FGF21 promotes endothelial cell function.

Fig 6. FGF21 promotes angiogenic response through the eNOS-dependent pathway.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Masuko Ushio-Fukai

Roles

Author response to Decision Letter 0

Decision Letter 1

Masuko Ushio-Fukai

Roles

Author response to Decision Letter 1

Decision Letter 2

Masuko Ushio-Fukai

Roles

Author response to Decision Letter 2

Decision Letter 3

Masuko Ushio-Fukai

Roles

Acceptance letter

Masuko Ushio-Fukai

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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