Dysregulation of ghrelin in diabetes impairs the vascular reparative response to hindlimb ischemia in a mouse model; clinical relevance to peripheral artery disease.

Type 2 diabetes is a prominent risk factor for peripheral artery disease (PAD). Yet, the mechanistic link between diabetes and PAD remains unclear. This study proposes that dysregulation of the endogenous hormone ghrelin, a potent modulator of vascular function, underpins the causal link between diabetes and PAD. Moreover, this study aimed to demonstrate the therapeutic potential of exogenous ghrelin in a diabetic mouse model of PAD. Standard ELISA analysis was used to quantify and compare circulating levels of ghrelin between (i) human diabetic patients with or without PAD (clinic) and (ii) db/db diabetic and non-diabetic mice (lab). Db/db mice underwent unilateral hindlimb ischaemia (HLI) for 14 days and treated with or without exogenous ghrelin (150 µg/kg/day.) Subsequently vascular reparation, angiogenesis, hindlimb perfusion, structure and function were assessed using laser Doppler imaging, micro-CT, microangiography, and protein and micro-RNA (miRNA) analysis. We further examined hindlimb perfusion recovery of ghrelin KO mice to determine whether an impaired vascular response to HLI is linked to ghrelin dysregulation in diabetes. Patients with PAD, with or without diabetes, had significantly lower circulating levels of endogenous ghrelin, compared to healthy individuals. Diabetic db/db mice had ghrelin levels that were only 7% of non-diabetic mice. The vascular reparative capacity of diabetic db/db mice in response to HLI was impaired compared to non-diabetic mice and, importantly, comparable to ghrelin KO mice. Daily therapeutic treatment of db/db mice with ghrelin for 14 days post HLI, stimulated angiogenesis, and improved skeletal muscle architecture and cell survival, which was associated with an increase in pro-angiogenic miRNAs-126 and -132. These findings unmask an important role for endogenous ghrelin in vascular repair following limb ischemia, which appears to be downregulated in diabetic patients. Moreover, these results implicate exogenous ghrelin as a potential novel therapy to enhance perfusion in patients with lower limb PAD, especially in diabetics.

Introduction

Type 2 diabetes mellitus (T2DM) is now a global pandemic that shows no sign of abatement. Diabetic patients have a greater predisposition to cardiovascular disease, in particular, T2DM is one of the most significant risk factors for lower limb peripheral artery disease (PAD). PAD is the progressive narrowing or occlusion of arteries as a result of atherosclerosis[1], which effectively increases the risk of mortality[2]. In 2015, it was estimated that over 236 million adults worldwide were living with PAD[3]. Importantly, PAD is one of the most common cardiovascular manifestations seen in T2DM patients[4]. A primary treatment for patients with severe PAD is surgical and endovascular revascularization[2]. However, despite considerable advances in surgical treatment, not all diabetic PAD patients are suited for surgical interventions[5]. In the clinic, ‘therapeutic angiogenesis’ remains a major challenge, as the use of pro-angiogenic agents or gene therapy have failed to translate to routine use in patients with PAD or diabetic PAD.

Recent studies indicate that the peptide hormone ghrelin may have a significant angiogenic role in PAD[6]. Ghrelin, first discovered in 1999[7], circulates in two distinct forms: acylated ghrelin (AG) and des-acylated ghrelin (DAG). Exogenous ghrelin has been shown to have an angiogenic potential in vitro[8,9] and in vivo[10-12], whilst also protecting against apoptosis[13] and inflammation[14], all of which are highly desirable mechanisms for the treatment of PAD. This is particularly important for patients with T2DM since decreased circulating levels of ghrelin is significantly associated with abdominal adiposity and insulin resistance[15].

We have previously shown that exogenous AG induces functional therapeutic angiogenesis in a young non-diabetic murine model of PAD, which was associated with the activation of key pro-angiogenic microRNAs (miRNAs)[11]. miRNAs are endogenous, small, non-coding ribonucleic acids of ~ 20–22 nucleotides which regulate gene expression at the post-transcriptional level, by either translational suppression or by mRNA degradation[16].

Here, we first aimed to conduct a small clinical study to investigate whether endogenous ghrelin is dysregulated in humans with PAD, with or without accompanying T2DM. Subsequently, we aimed to determine whether the vascular reparative response to hindlimb ischemia is impaired in diabetic db/db mice and, elucidate the role of endogenous ghrelin in driving the vascular reparative response to ischemia using ghrelin knock out mice. Finally, we aimed to ascertain the therapeutic potential of exogenous ghrelin for promoting functional angiogenesis in an aged, T2DM murine model of PAD.

Materials and methods

A detailed description of the methods is provided in the online ‘Supplementary Methods’ file.

Clinical study: plasma ghrelin in human PAD patients with or without diabetes

Four cohorts of patients were studied: (1) PAD patients with T2DM (PAD + T2DM), (2) PAD patients without T2DM (PAD), (3) T2DM patients with no PAD (T2DM), and (4) control subjects with no T2DM or PAD (Control). Ethical approval was obtained from the University of Otago Human Ethics Committee (Health) (H17/012), and the study conformed to the standards set by the Declaration of Helsinki. All participants were 50 years of age or older, and all provided informed written consent. Participant demographics are presented in Table 1.

Table 1

Participant demographics.

	Controls(n = 30)	T2DM(n = 25)	PAD(n = 41)	PAD + T2D(n = 26)
Males (%)	17 (57)	12 (48)	30 (73)	18 (69)
Age (y)	61.1 ± 6.7	63.6 ± 8.2	71.9 ± 10.7 *†	72.8 ± 9.0 *†
BMI (kg/m2)	28.3 ± 5.1	32.3 ± 7.2	25.9 ± 5.1 †	29.3 ± 5.9
HbA1c (mmol/mol)	35 (33–37)	64 (51–77) *	38 (35–41) *‡	57 (45–63) *
Resting ABI in lower limb	1.1 ± 0.1	1.1 ± 0.1	0.6 ± 0.2 *†	0.6 ± 0.3 *†
Smoking status: n = current/ex/never	1/8/21	0/10/15	17/17/7	3/19/4

Age, BMI and ABI are shown as mean ± SD; HbA1C, is shown as median (IQR); The categorical data is presented as number (percentage).

T2DM type 2 diabetes mellitus, PAD peripheral arterial disease, BMI body mass index, HbA1c glycosylated haemoglobin, ABI ankle-brachial index.

*P < 0.05 versus control group.

†P < 0.05 versus T2DM group.

‡P < 0.05 versus PAD group.

A venous blood sample was drawn, centrifuged, snap-frozen and stored at − 80 °C. Human acylated ghrelin (AG) and des-acylated ghrelin (DAG) were quantified as previously described[17] using the easy sampling enzyme immunoassay kits purchased from Bertin Pharma (Montigny-le-Bretonneux, France; Cat No: A05306 and A05319 respectively).

Experimental study: role of ghrelin in vascular reparation following hindlimb ischemia

Animals

All animal experimentation was approved by the Animal Ethics Committee of the University of Otago, New Zealand, and the National Cerebral and Cardiovascular Centre, Japan. All procedures are reported in accordance with the ARRIVE guidelines. Diabetic db/db (DM; DM, BKS.CG-DOCK7+/+Lepr/J) and non-diabetic (ND; db/+) littermates were obtained from Jacksons Laboratories, Japan, or bred at the Hercus-Taieri breeding facility at the University of Otago.

Ghrelin knock out mice (KO—knockout of the preproghrelin gene), and their wildtype littermates (WT) were bred at the Hercus-Taieri breeding facility (New Zealand) or the National Cerebral and Cardiovascular Centre (Japan).

Hindlimb ischemia (HLI)

Using standard aseptic procedures, unilateral limb ischemia of the upper left femoral artery was induced by dual ligation of the femoral artery (with a 6-0 silk suture) at two distinct points as well as electrocoagulation as previously described[11].

Laser Doppler imaging

Hindlimb blood flow was sequentially analysed 1 h and then again at 3, 7, 10, and 14 days post-surgery in the ischemic and contralateral feet. Hindlimb flow was measured using a real-time microcirculation imager (PeriCam PSI HR system, Perimed, Sweden), which operates on the Laser Speckle Contrast Analysis Technology (LASCA) and measures blood perfusion in arbitrary Perfusion Units (PU).

Microangiography

Vascular function and hindlimb perfusion were assessed and quantified using Synchrotron microangiography as previously described[18].

Protocol

Mice were positioned supine above the X-ray source, and a single bolus of contrast agent was injected at high speed (0.1 mL @ 0.4 mL/s) into the iliac artery for imaging of left hindlimb vessels. Following baseline imaging, hindlimb angiograms were recorded in response to: (i) acetylcholine (ACh, 10 µg/kg/min for 5 min, i.v) to assess endothelium-dependent vasodilation, (ii) the NO donor sodium nitroprusside (SNP, 5 µg/kg/min for 5 min, i.v.) to assess endothelium-independent vasodilation and iii) the Rho-Kinase inhibitor Fasudil (20 mg/kg intravenous bolus).

Immunohistochemistry and histological analysis

For analysis of capillary and arteriole density, gastrocnemius muscle sections (7 μm) were stained with isolectin-B4 (Vector Laboratories, B-1205; 1:100) to identify endothelial cells and the smooth muscle marker, α-smooth muscle actin conjugated to Cy3 (Sigma-Aldrich, C6198, 1:100) as previously described[19]. High resolution images were acquired (at × 200) and counts from 18 randomly selected fields were averaged and expressed as the number of capillaries and arterioles (< 50 μm) per mm2 of muscular sections[20].

DNA fragmentation, associated with apoptosis, was detected in sections (7 μm) of gastrocnemius muscle using the commercially available terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) kit (Click-iT Plus TUNEL Alexa Fluor 594 kit, Life Technologies). Sections were counterstained with Isolectin B4 to identify endothelial cells and DAPI to label nuclei.

Skeletal tissue fibrosis was detected in transverse sections (7 µm) of gastrocnemius muscle using Mason’s Trichrome staining.

RNA isolation and quantitative real-time PCR

Total RNA was isolated from ischemic gastrocnemius tissue samples using the miRNeasy mini kit (Qiagen). Real-time PCR was performed with Applied Biosystems miRNA assays (miRNAs -126, 132, -206, and -92a) on a StepOnePlus device.

Western blot analyses

Ischemic gastrocnemius tissue was homogenised and total protein (20 µg) was resolved with SDS-PAGE, transferred onto a PVDF membrane, and probed with the following antibodies: VEGFA (SantaCruz Biotechnology, sc-152; 1:1,000), BCL-2 (Cell Signaling, 1:1,000), AKT (Cell Signaling, 1:1,000), and Phospho-AKTser473 (Cell Signaling, 1:1,000). For detection, goat anti-rabbit secondary (Abcam; 1:2000) conjugated to horseradish peroxidase was used, followed by chemiluminescence reaction with clarity ESC blotting substrate (Bio-Rad).

Mouse ELISA

Blood (0.2 mL) was collected into a EDTA microvette, stabilised with 4-(2-aminoethyl) benzenesulphonyl fluoride (1:100; A8456 Sigma-Aldrich)[17], centrifuged (< 1 min) at 1,500×g, and aliquoted. AG concentrations were quantified using the AG (mouse, rat) easy sampling EIA Kit (Bertin Pharma) according to the manufacturer’s instructions.

Volumetric microcomputer tomography and whole-mount imaging

Hindlimb angioarchitecture was assessed in separate cohorts of mice for each experimental group using microcomputer tomography (micro-CT) analysis. The lower half of the mouse was perfused with Microfil (MV122-Yellow; Flow Tech Inc) scanned on a SkyScan-1172 high-resolution micro-CT system (Skyscan, Aarteselar, Belgium) at a 17.4 μm resolution, a voltage of 60 kV, and a current of 167 μA.

Results

Downregulation of ghrelin in patients with peripheral artery disease and type 2 diabetes

Circulating levels of ghrelin were analyzed in plasma samples collected from patients with T2DM + PAD, non-diabetic PAD, and their respective controls (non-PAD). Patient characteristics are presented in Table 1. The population comprised equal proportions of male vs female (P = 0.15), although age did differ between groups, with PAD and PAD + T2DM participants slightly older than their non-PAD counterparts (P < 0.002). HbA1c and ABI confirmed group allocations with respect to diabetes status and PAD.

Plasma AG in all diseased groups was significantly lower than that of the non-diabetic, non-PAD control subjects (P = 0.049, see Supplementary Fig. S1 & Table 2), although Dunn’s correction for multiple comparisons revealed no significant differences in AG between any of the diseased groups. The ROC curves used to determine the threshold value for plasma AG to distinguish diseased groups from controls was 67 pg/mL (Table 3). Subsequent logistic regression analysis revealed a significant association between AG < 67 pg/mL and having T2DM, PAD and PAD + T2DM (Table 4).

Table 2

Ghrelin parameters.

	Controls (n = 30)	T2DM (n = 25)	PAD (n = 41)	PAD + T2D (n = 26)
AG (pg/mL) *	87 (70–136)	46 (36–96)	70 (28–114)	46 (25–116)
DAG (pg/mL)	116 (71–129)	67 (46–103)	92 (47–155)	96 (29–176)
AG:DAG ratio	0.90 (0.74–1.11)	0.84 (0.52–1.16)	0.74 (0.36–1.43)	0.64 (0.37–1.20)
Total G (pg/mL)	209 (145–695)	126 (87–202)	175 (85–251)	147 (59–334)

T2DM type 2 diabetes mellitus, PAD peripheral arterial disease, AG acylated ghrelin, DAG deacylated ghrelin, Total G, total ghrelin. AG, DAG, AG:DAG ratio and Total G are shown as median (IQR).

*P = 0.049 for a difference between groups.

Table 3

Cut off values calculated using the Youden index from the receiver operating characteristic (ROC) curve.

Group	Cut-off value (pg/mL)	Sensitivity (%)	Specificity (%)	AUROC	AUROC 95% CI
T2D	75	72	73	0.71	0.57–0.82
PAD	67	49	80	0.63	0.51–0.75
PAD + T2DM	76	69	73	0.68	0.54–0.80

T2DM type 2 diabetes mellitus, PAD peripheral arterial disease, AUROC area under the ROC curve.

Table 4

Logistic regression analysis to calculate the odds ratios for having acylated ghrelin (AG) below the threshold 67 pg/mL.

Group	Unadjusted odds ratio			Adjusted for age
	Odds ratio	95% CI	p-value	Odds ratio	95% CI	p-value
T2DM: AG < 67 pg/mL	6.00	1.81–19.93	0.003	5.82	1.74–19.42	0.004
PAD: AG < 67 pg/mL	3.46	1.17–10.22	0.025	2.92	0.90–9.48	0.075
PAD + T2DM: AG < 67 pg/mL	6.40	1.94–21.11	0.002	5.32	11.47–19.12	0.011

The reference population was the control group. The adjusted model includes age. Adjusting for sex had no effect (data not shown)

T2DM type 2 diabetes mellitus, PAD peripheral arterial disease.

AG expression has been suggested to decline with age in males[21]. Adjusting the model for age, the significant association remained. Moreover, there was no significant correlation between ‘age’ and ‘plasma AG’ in any of the groups (P = 0.24 for all groups). Similarly, there was no significant association between HbA1c and AG in any of the groups (P = 0.15 for all groups).

Reduced ghrelin levels are associated with impaired vascular reparation following HLI

To determine if reduced ghrelin levels are associate with impaired recovery of vascular perfusion after HLI, we induced HLI in aged diabetic db/db mice.

Circulating ghrelin levels are reduced in diabetic db/db mice

The plasma AG concentration in db/db diabetic mice (DM) prior to HLI (44 ± 14 pg/mL) was only 7% of the non-diabetic (ND) mice (593 ± 157 pg/mL; Fig. 1A). Moreover, circulating AG in ND mice progressively increased over 14 days of HLI (P < 0.01), whereas AG did not change following HLI in DM mice.

Figure 1

Circulating acylated ghrelin (AG) is reduced in diabetic db/db mice. (A) A comparison of plasma AG between non-diabetic (ND) and diabetic (DM) mice before and after 14 days of hindlimb ischemia (HLI; n = 6). (B) Representative laser Doppler images of ND and DM mice 1 h after HLI (Day 0), and then 7 and 14 days after HLI. Red squares delimit the area measured in the ischemic foot. Data are quantified in the adjoining line graph showing the time course of blood flow recovery (calculated as the ratio of blood flow in ischemic to contralateral foot; n = 5–6). (C) Representative micro-CT images showing the hindlimb vasculature in ND and DM mice after 14 days of HLI. The red oval indicates the region of interest for quantifying vascular volume, as presented in the adjoining bar graph (n = 5–6 for all study groups). (D) Representative ischemic gastrocnemius sections stained with the endothelial marker isolectin B4 (green fluorescent) and a-smooth muscle actin (red fluorescent) in the arterial wall (scale bar, 50 μm) of ND and DM mice 14 days after HLI. Both capillary and arteriole density (diameter < 50 μm) are quantified in the adjoining bar graphs (n = 5–6). Data are presented as mean ± S.E.M. Statistical comparisons were made using two-way ANOVA, followed by Sidak multiple comparisons post hoc tests, or unpaired t-tests. *Significant difference between ND and DM (**P < 0.01, ***P < 0.001); ||Significant difference between Sham and HLI (|| || ||P < 0.001).

Hindlimb revascularization and vascular reparation following HLI is impaired in diabetes

Laser Doppler perfusion imaging revealed that the initial ischemic insult to the hindlimb following HLI was more severe in DM mice compared to ND mice (ischemic to non-ischemic hindlimb perfusion ratio of 0.2 and 0.34, respectively; Fig. 1B). Although there was a time-dependent restoration of hindlimb blood flow in both ND and DM mice, limb perfusion in DM mice never improved to the same level as seen in the ND mice (Fig. 1B).

At day 14 post-HLI, hindlimb vasculature volume, which comprises the enlarged collateral vessels (micro-CT; Fig. 1C), as well as capillary and arteriole (< 50 µm) densities (immunohistochemistry; Fig. 1D) were significantly lower in DM mice compared with ND mice.

Specific role of endogenous ghrelin in hindlimb revascularization following HLI

To selectively identify whether dysregulation of endogenous ghrelin in diabetes underpins the impaired revascularization response following HLI, we quantified the vascular reparative response in ghrelin KO mice, which lack other confounding effects associated with diabetes.

Ghrelin knockout impairs post-HLI revascularization

Similar to that previously observed for the diabetic db/db mice, ghrelin KO mice had a poorer recovery in hind limb blood flow after 14 days post-HLI (Fig. 2A,B), reduced vascular volume (micro-CT; Fig. 2C–E) and reduced capillary/arteriole density (Fig. 2F–H), compared to the wild type counterpart. Consequently, the functional capacity of the hindlimb was impaired in KO mice (Supplementary Fig. S2).

Figure 2

Restoration of blood flow after hindlimb ischemia is impaired in ghrelin knockout mice. (A) Representative laser Doppler images at baseline, day 7, and day 14 following HLI in KO and WT mice. Red squares delimit the area measured in the ischemic foot. (B) Time course of blood flow recovery calculated as the ratio of blood flow in ischemic to contralateral foot (n = 7–10). (C) Representative micro-CT images are showing the hindlimb vasculature in KO and WT mice undergoing HLI for 14 days. (D) Quantification of vascular volume by micro-CT analysis (n = 4–6). (E) Representative whole-mount images of Microfil vascular cast limbs. White arrow heads indicate enlarged arteries, pink arrow heads indicate ligations. (F) Representative ischemic gastrocnemius sections stained with the endothelial marker, isolectin-B4 (green fluorescent) and the smooth muscle marker, α-smooth muscle actin (red fluorescent). Scale bar, 50 μm. Quantification of (G) capillary and (H) arteriole density (diameter < 50 μm) density at day 14 post HLI (n = 5–7). Data are presented as mean ± S.E.M. Statistical comparisons were made by using a RM two-way ANOVA, followed by Sidak multiple comparisons test, (B) two-tailed unpaired Student t test (E, G), or two-tailed unpaired Mann–Whitney U test (H). *Significantly different from WT counterpart (*P < 0.05, **P < 0.01, ***P < 0.001).

Microangiographic assessment of vascular function: role of endogenous ghrelin

Changes in hindlimb vascular function following HLI was assessed based on the vasodilatory responses to ACh (endothelial-dependent) and SNP (endothelial-independent) using microangiography (Supplementary Fig. S3A-B). In sham mice, the vasodilatory responses of all measured vessels to both ACh and SNP were not significantly different between WT and KO, indicating normal vascular function in KO sham mice (Supplementary Fig. S3C,D). However, following 14 days of HLI, the vasodilatory responses to ACh and SNP, especially the 50–100 µm vessels, were significantly blunted in KO mice, reflecting impaired vascular function (Supplementary Fig. S3C,D).

The mean arterial blood pressure (MABP) and heart rate (HR) responses to ACh and SNP were mostly similar across all groups (Supplementary Fig. S4).

Ghrelin knockout reduces tissue repair and pro-angiogenic miRNAs following HLI

HLI was associated with an increase in TUNEL+ apoptotic cell (Fig. 3A,C) and endothelial cell counts (Fig. 3B,D) in the gastrocnemius muscle, which was significantly higher in KO mice by 154% and 446% respectively, compared to WT mice (Fig. 3A,C). In addition, the severity of fibrosis following HLI was amplified in KO mice compared to WT mice (Fig. 3E,F).

Figure 3

Deficiency of endogenous ghrelin promotes apoptosis and fibrosis after hind limb ischemia. (A) Representative images of TUNEL-positive myocytes and (B) endothelial cells in the ischemic gastrocnemius muscle, 14 days post-HLI (C) Quantification of TUNEL-positive myocytes and (D) endothelial cells represented per mm2 (n = 4–5). Scale bars = 50 μm (Inset scale bar = 25 µm). (E) Representative histological images of Masson’s trichrome staining in the ischemic gastrocnemius muscle of WT and KO mice 14 days post-HLI. (F) Quantification of gastrocnemius muscle fibrosis (n = 5–6). Scale bars = 200 μm. Data are presented as mean ± S.E.M. Statistical comparisons were made by using a two-tailed unpaired Mann–Whitney U test (C,D,F). *Significantly different from WT counterpart (***P < 0.001).

Consistent with the observed lack of angiogenesis and arteriogenesis in KO mice following 14 days of HLI, the expression of pro-angiogenic miRNAs -126 and -132 (Supplementary Fig. S5A,B), as well as the downstream angiogenic protein VEGF (Supplementary Fig. S5E), were significantly lower in the ischemic hindlimb of KO mice compared to that of WT mice. However, the anti-angiogenic miRNAs -92a and -206 (Supplementary Fig. S5C-D), and the pro-survival proteins BCL-2 and pAKT (Supplementary Fig. S5F, G), were not different between KO and WT mice.

Role of exogenous ghrelin for improving the revascularization response to HLI in diabetes

Having confirmed the significant role of endogenous ghrelin for driving the revascularization response following HLI, and considering that circulating levels of ghrelin are adversely reduced in diabetic mice, we next tested the efficacy of exogenous ghrelin (both AG and DAG) to promote revascularization and angiogenesis in diabetic db/db mice with HLI.

Recovery of hind limb blood flow following HLI in diabetes: ghrelin therapy

The daily administration of AG to both DM and ND mice significantly improved perfusion recovery over the 14 days post-HLI, such that laser Doppler imaging showed that limb perfusion of treated DM mice was similar to ND mice (Fig. 4A,B). However, DAG did not significantly improve blood flow recovery at day 14 in DM mice, although it did improve recovery in ND mice.

Figure 4

Exogenous ghrelin enhances tissue perfusion after hind limb ischemia in diabetic db/db mice. (A) Representative laser Doppler images of ND and DM mice undergoing HLI and treated with vehicle, AG, or DAG for 14 days. Red squares delimit the area measured in the ischemic foot. (B) Time course of blood flow recovery (calculated as the ratio of blood flow in ischemic to contralateral foot; n = 5–6). (C) Representative micro-CT images showing the hindlimb vasculature in ND and DM mice undergoing HLI and administered with either vehicle, AG, or DAG for 14 days. The red oval indicates the measured area region of interest. (D) Quantification of vascular volume by micro-CT analysis among all study groups (n = 5–6). (E) Representative ischemic gastrocnemius sections stained with the endothelial marker isolectin B4 (green fluorescent) and a-smooth muscle actin (red fluorescent) to identify smooth muscle cells in the arterial wall (scale bar, 50 μm). (F) Quantification of the capillary and (G) arteriole density (diameter < 50 μm) density in ischemic gastrocnemius of mice at 14 days post ischemia (n = 5–6). Data are presented as mean ± S.E.M. Statistical comparisons were made using a RM two-way ANOVA, followed by Tukey’s multiple comparisons post hoc tests. †Significantly different from ND Veh (†P < 0.05, ††P < 0.01, †††P < 0.001); §Significantly different from ND DAG (§P < 0.05, §§§P < 0.001); ||Significantly different from DM Sham (|| ||P < 0.01, || || ||P < 0.001); #Significantly different from DM Veh (#P < 0.05, ##P < 0.01, ###P < 0.001); ¥Significantly different from DM DAG (¥P < 0.05, ¥¥¥P < 0.001).

AG treatment to both ND and DM mice significantly augmented the increase in vascular volume associated with HLI, compared to vehicle alone (micro-CT; Fig. 4C,D), as well as augmenting the increase in capillary and arteriole density (α-smooth muscle actin staining; Fig. 4E–G). Although DAG increased vascular volume (Fig. 4C,D) and arteriole density above vehicle alone (Fig. 4G), it did not enhance capillary density in ND and DM mice (Fig. 4F).

Collectively, the actions of AG in diabetes to improve blood perfusion, vascular volume, and capillary/arteriole formation in the ischemic hindlimb, likely underpins the improved functional capacity compared to untreated DM mice (Supplementary Fig. S6).

Ghrelin therapy improves vascular function during diabetic HLI

Diabetes was associated with impaired vascular function within the ischemic limb following HLI (Supplementary Fig. S7). We next assessed the ability of ghrelin (AG and DAG) to preserve vascular function in diabetic db/db mice following HLI using micoangiography. The vasodilatory response of the 50–150 µm vessels to ACh was impaired by HLI, diabetes or the combination of both (Supplementary Fig. S7B). Remarkably, AG treatment in DM + HLI mice improved the vascular response to ACh so that it was essentially indistinguishable from control mice (i.e. ND + Sham; ~ 40% increase in ID of the 50–100 µm vessels; Supplementary Fig. S7B). In comparison, DAG did not improve the blunted responses to ACh, greater than that of vehicle alone, in either the DM or ND mice. The vasodilatory response to SNP did not appear to be compromised by either HLI or diabetes, such that the magnitude of vasodilation was comparable between all ND and DM groups (Supplementary Fig. S7C).

The vasodilatory responses to Rho-kinase inhibition (Fasudil), particularly of the 50–150 µm sized vessels, appeared to be exacerbated only in diabetic mice with or without accompanying HLI (40 ± 7% increase in ID for the 50–100 µm vessels; Supplementary Fig. S7D). Importantly, AG administration to diabetic mice normalized the vasodilatory response to Fasudil, similar to ND + Sham mice (Supplementary Fig. S7D).

Ghrelin therapy enhances cell survival and decreases fibrosis

Diabetes was associated with a significant increase in both total TUNEL+ apoptotic cells and TUNEL+ endothelial cells (TUNEL and isolectin-B4 positive; Fig. 5A–D), which was further exacerbated following 14 days of HLI. AG therapy was able to significantly reduce the adverse increase in total TUNEL+ apoptotic and TUNEL+ endothelial cells, particularly in DM mice (Fig. 5A–D). In comparison, DAG did not attenuate the increase in total TUNEL+ apoptotic cells (Fig. 5C), but it did attenuate the increase in TUNEL+ endothelial cells (Fig. 5D).

Figure 5

Exogenous ghrelin aids cell survival after hind limb ischemia in diabetic db/db mice. (A) Representative images of TUNEL-positive nuclei (blue = nuclei) and TUNEL-positive endothelial cells (green = endothelial) in the gastrocnemius muscle 14 days post ischemia; scale bars = 50 μm. (B) An inset is magnified to highlight co-localisation of tunnel stained nuclei or endothelial cells; scale bar = 15 µm. (C) Quantification of TUNEL-positive nuclei per mm2 (n = 4–6) and (D) Quantification of TUNEL-positive endothelial cells per mm2 (n = 4–6). (E) Representative histological images of Masson’s trichrome staining among all study groups following 14 days of HLI; Scale bar = 50 μm. (F) Quantification of gastrocnemius muscle fibrosis (n = 4–7). Data are presented as mean ± S.E.M. Statistical comparisons were made using a two-way ANOVA, followed by Tukey post hoc test (B,D,F). *Significantly different from ND Sham (***P < 0.001); †Significantly different from ND Veh (†P < 0.05, †††P < 0.001); ||Significantly different from DM Sham (||P < 0.05, || || ||P < 0.001); #Significantly different from DM Veh (##P < 0.01, ###P < 0.001); ¥Significantly different from DM DAG (¥¥P < 0.01).

HLI-induced injury was also associated with muscle collagen deposition, i.e. fibrosis (Fig. 5E), which was significantly worse in DM mice compared to ND mice (Fig. 5F). Treatment with AG and DAG was effective in reducing the severity of HLI-induced fibrosis in DM and ND mice, although the anti-fibrotic effects were more pronounced with AG compared to that of DAG (Fig. 5F).

Ghrelin treatment activates pro-angiogenic miRNAs

In ND mice, both AG and DAG treatment following HLI significantly increased the expression of pro-angiogenic miRNAs -126 and -132, compared to vehicle treatment, whilst also suppressing the HLI-induced overexpression of anti-angiogenic miRNAs-206 and -92a (Fig. 6A–D). Importantly, the effect of AG and DAG on miRNA expression was associated with the observed increase in the expression of VEGF, pAKT and Bcl-2 proteins (Fig. 6E–G).

Figure 6

Ghrelin treatment enhances pro-angiogenic and pro-survival miRNAs and proteins after hind limb ischemia in diabetic db/db mice. Bar graphs represent the average relative expression of (A) miRNAs-126, (B) 132, (C) 92a, and (D) 206 normalised to the small nuclear U6 snRNA, which was used as a normaliser, among the study groups at 14 days following HLI (n = 5–6). Representative western blot images and protein quantification of (E) VEGFa, (F) BCL-2, and (G) pAKT in the ischemic gastrocnemius muscle among the study groups at 14 days following HLI (n = 5–6). Ponceau-S staining was used as a loading control. Data are presented as mean ± S.E.M. Statistical comparisons were made using two-way ANOVA, followed by Tukey post hoc test (A through G). *Significantly different from ND Sham (*P < 0.05, **P < 0.01, ***P < 0.001); †Significantly different from ND Veh (†P < 0.05, ††P < 0.01, †††P < 0.001); ||Significantly different from DM Sham (||P < 0.05, || || ||P < 0.001); #Significantly different from DM Veh (#P < 0.05, ###P < 0.001); ¥Significantly different from DM DAG (¥P < 0.05, ¥¥P < 0.01).

In DM mice, the small increase in miRNA-126 and -132 expression following AG treatment was not significant, although AG did suppress over-expression of miRNA-206 (Fig. 6D) and, importantly, increased VEGF, pAKT and Bcl-2 protein expression (Fig. 6E–G). In contrast, DAG was ineffective in modulating any of the HLI-induced changes in miRNAs or the associated target proteins (Fig. 6A–G).

Discussion

Diabetes remains one of the greatest risk factors for the onset of PAD, which is associated with an unacceptably high premature mortality rate[22]. Restoring or increasing perfusion to alleviate ischemia has been the goal of clinical research for several decades. However, potentially promising experimental strategies for promoting functional angiogenesis have often failed to reach routine clinical application. Here, we provide novel data identifying the important role of endogenous ghrelin, which is dysregulated in T2DM, for driving the angiogenic response following ischemia to improve limb perfusion. Moreover, exogenous ghrelin treatment appears to improve revascularization, at least in the diabetic db/db mouse model, that may be mediated via the modulation of key vascular-specific miRNAs-126, -132, -92a, and -206.

Ghrelin is derived from preproghrelin, a 117 amino-acid precursor that is predominantly produced by P/D1-like cells in the oxyntic glands of the gastric mucosa[23]. Acylated ghrelin and its receptor (GHS-R) are ubiquitous throughout the cardiovascular system[23,24], suggesting ghrelin has a prominent role in cardiovascular homeostasis[25]. To date, a growing body of literature advocates the beneficial effects of AG in maintaining cardiovascular health[6,26,27].

Clinically, endogenous levels of ghrelin appear to be downregulated in diabetic patients[15], an observation confirmed in this study. Herein, we further show that both diabetic and non-diabetic patients with PAD had low circulating levels of acylated ghrelin (below a critical level of < 67 pg/mL). These results may suggest that dysregulation of endogenous ghrelin, rather than diabetes per se, may be the crucial risk factor for developing PAD. Hence, low plasma levels of endogenous AG in the clinic may serve at the very least as a prognostic marker for PAD progression.

Experimental data in this study showed that the magnitude of damage to the hindlimb microvasculature following prolonged ischemia was essentially the same for diabetic db/db mice, which had only ~ 7% on the circulating ghrelin levels compared to non-diabetic mice, and ghrelin knock out mice, which lacked diabetes. These observations once again strengthen the idea that dysregulation of endogenous ghrelin, which is a consequence of diabetes, underpins the development of PAD.

Role of miRNAs for mediating angiogenesis following HLI

The mechanism(s) by which ghrelin dysregulation underpins impaired vascular reparation and angiogenesis remains unclear. We have previously reported that the expression of pro-angiogenic miRNAs-126 and -132, which were correlated to circulating ghrelin levels, appeared to determine the severity of hindlimb vascular damage following HLI, at least in a young, healthy mouse model[11]. Togliatto et al. also reported that increasing circulating levels of des-acylated ghrelin promoted skeletal muscle regeneration via the modulation of miRNAs-221 & -222[28], and protected the hindlimb vasculature against reactive oxygen species-mediated damage, through the modulation of miRNA–126[29].

In this study, we aimed to more closely emulate the clinical setting and, thus, report the efficacy of acylated ghrelin treatment in an aged, diabetic murine model of PAD. We report that the downregulation of miRNA-126 and -132, in both the diabetic and ghrelin KO mouse model mirrored the level of endogenous ghrelin and importantly, was closely associated with the impaired vascular reparative ability following HLI. The protein targets for miRNA–126, SPRED1 and PIK3R2, are both negative regulators of VEGFA[30]. Moreover, miRNA-132 targets p120RasGap and Ras-activation, which are known inhibitors of angiogenesis[31]. Hence, it is mechanistically plausible that the modulation of select miRNAs may underpin the pathway by which ghrelin facilitates its vascular-protective effects, independent of diabetes, although further mechanistic studies are essential to confirm this hypothesis.

Therapeutic potential of exogenous ghrelin for HLI

In this study, we observed that daily AG administration following HLI in diabetic db/db mice dramatically improved hindlimb blood flow perfusion recovery, through improved vascular function, increased cell survival, angiogenesis/arteriogenesis, and skeletal tissue architecture recovery. Interestingly, DAG also exhibited some beneficial effects but consistently to a lesser extent than AG treatment. We also observed that these beneficial vascular effects driven by AG were associated with modulation of specific miRNAs. These results once again suggest that ghrelin may elicit its vascular effects, at least in part, through the modulation of miRNAs.

Arpino et al.[32] demonstrated that vascular regeneration following HLI constitutes a highly flawed vascular network, with an impaired vasomotor control system. In agreement, we were able to show, with the use of high-resolution microangiography, that the impaired vascular regeneration response to HLI observed in ghrelin KO mice and diabetic db/db mice was associated with vascular endothelial dysfunction, based on the blunted vasodilatory responses to ACh. Remarkably, however, ghrelin therapy to diabetic mice not only amplified the angiogenic response to HLI, but endothelial function of these newly formed vessels was functionally normal (compared to ND sham animals).

The hindlimb vasculature of diabetic sham mice had a blunted vasodilatory response to ACh, which we have also recently demonstrated in the coronary vasculature of diabetic db/db mice[33]. Importantly, considering that ghrelin treatment to diabetic sham animals restored vascular function to a level that was indistinguishable from their ND counterparts, it is likely that dysregulation of ghrelin in diabetes underpins the observed endothelial dysfunction in diabetes.

Conclusion

In summary, endogenous ghrelin plays an important role in driving the crucial revascularization response following ischemia. Clinically, human patients with diabetes with or without PAD appear to have reduced levels of AG. We also demonstrate that exogenous AG treatment in a diabetic murine model of PAD promotes functional vascularization, likely through the modulation of specific angiogenic miRNAs. Ultimately, this study highlights the future potential of exogenous AG as a novel therapeutic intervention for the adjunct treatment of PAD in both diabetic and non-diabetic patients.