β 3 Adrenergic Receptor Stimulation Promotes Reperfusion in Ischemic Limbs in a Murine Diabetic Model

Abstract

Aims/Hypothesis: Peripheral arterial disease (PAD) is a major burden, resulting in limb claudication, repeated surgical interventions and amputation. There is an unmet need for improved medical management of PAD that improves quality of life, maintains activities of daily life and reduces complications. Nitric oxide (NO)/redox balance is a key regulator of angiogenesis. We have previously shown beneficial effects of a β ₃ adrenergic receptor (β ₃AR) agonist on NO/redox balance. We hypothesized that β ₃AR stimulation would have therapeutic potential in PAD by promoting limb angiogenesis. Methods: The effect of the β ₃AR agonist CL 316,243 (1-1,000 nmol/L in vitro, 1 mg/kg/day s. c) was tested in established angiogenesis assays with human endothelial cells and patient-derived endothelial colony forming cells. Post-ischemia reperfusion was determined in streptozotocin and/or high fat diet-induced diabetic and non-diabetic mice in vivo using the hind limb ischemia model. Results: CL 316,243 caused accelerated recovery from hind limb ischemia in non-diabetic and type 1 and 2 diabetic mice. Increased eNOS activity and decreased superoxide generation were detected in hind limb ischemia calf muscle from CL 316, 243 treated mice vs. controls. The protective effect of CL 316,243 in diabetic mice was associated with >50% decreases in eNOS glutathionylation and nitrotyrosine levels. The β ₃AR agonist directly promoted angiogenesis in endothelial cells in vitro. These pro-angiogenic effects were β ₃AR and NOS-dependent. Conclusion/Interpretation: β ₃AR stimulation increased angiogenesis in diabetic ischemic limbs, with demonstrable improvements in NO/redox balance and angiogenesis elicited by a selective agonist. The orally available β ₃AR agonist, Mirabegron, used for overactive bladder syndrome, makes translation to a clinical trial by repurposing of a β ₃AR agonist to target PAD immediately feasible.

Keywords: hind limb ischemia; nitric oxide; peripheral artery disease; redox; vascular.

FIGURE 1

β₃AR stimulation promotes angiogenesis in vitro. (A) cell migration by scratch assay over 24 h in response to increasing concentrations of CL 316,243 in HUVECs. ×4 magnification of 96 well plate.; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs vehicle control by 2-way ANOVA and (B) tubule formation in HUVECs grown on reduced growth factor Cultrex extracellular matrix, in response to increasing concentrations of β3 AR agonist CL 316,243. *p < 0.05, **p < 0.01, vs vehicle control by 1-way ANOVA with Bonferroni post-hoc analysis, n = 6 from 3 experiments. All data shown is mean ± SEM. Representative images depict data obtained from vehicle control and CL 316,243-treated (100 ng/ml) cells, with panel A showing closure at baseline (left) and after 15 h (right).

FIGURE 2

β₃AR-stimulated angiogenesis is NOS-dependent. (A) Tubule formation in HUVECs and the effect of l-NAME (300 μmol/L); n = 5 (B) cell migration rate in HUVECs and the effect of l-NAME (300 μmol/L); n = 4. (C) Tubule formation in human adult dermal microvascular endothelial cells (MVEC) and (D) patient-derived endothelial colony forming cells (ECFC) with β ₃AR agonist CL 316,243 (100 ng/ml) and β ₃AR antagonist, SR 592630 A (1 μmol/L) n = 3. (E) Representative images in ECFCs and (F) participant characteristics and medical history of ECFC source. Mean ± SEM; **p < 0.01, ****p < 0.0001 vs control; ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 vs. CL 316,243 by 1-way ANOVA with Bonferroni post-hoc analysis or 2-way ANOVA. ACE/ARB, angiotensin converting enzyme inhibitor/angiotensin receptor blocker; BMI, body mass index; CACS, coronary artery calcium score; SPS, soft plaque score.

FIGURE 3

β₃AR stimulation accelerates reperfusion following hind limb ischemia. (A) Representative images showing Laser Doppler flux in hindlimbs from mice immediately post-ligation and after 14 days of recovery, treated with vehicle (saline) or CL 316,243 (1 mg/kg/day) (B) Summary data of hind limb perfusion both pre- and post-ligation and in the contralateral control limb shown as raw flux data. (C) Calculated ratio of the ischemic to non-ischemic limbs following 14 days of hind limb ischemia. (D) eNOS activity by radioimmunoassay in hind limb tissue from mice treated with vehicle or CL 316,243, at 14 days post-surgery. (E) Superoxide generation measured by lucigenin-enhanced chemiluminescence (20 μmol/L) corrected for background luminescence. (F) Immunoblot expression of hindlimb Nox 2 and Nox 4 with β-actin control, V = vehicle, CL = CL-316,243-treated. Mean ± SEM; *p < 0.05 vs vehicle by 1-way or 2-way ANOVA with Bonferroni post-hoc analysis; n = 8. ⁺⁺⁺ p < 0.001 vs. Pre-ligation limb perfusion; ^### p < 0.001 vs. post-ligation (timepoint 0) reperfusion.

FIGURE 4

Effect of β3AR stimulation in hind limb ischemia, as measured by laser doppler imaging, in type 1 diabetic mice. (A) Schematic diagram showing the study protocol. (B) Representative image in type 1 diabetic mouse at the end of the study (day 28) and (C) representative image of CD31 staining (left). Right, ratio of perfusion in ischemic to non-ischemic limbs in citrate-buffer control (n = 7–8) and type 1 diabetes (T1D, n = 10) mice treated with vehicle (saline) or CL 316,243 (1 mg/kg/day, s. c. 28 days). Mean ± SEM; *p < 0.05, **p < 0.01 ***p < 0.001 vs. vehicle by 2-way ANOVA with Bonferroni post-hoc analysis. (C) CD31 expression in hind limbs post-ischemia in control and diabetic mice treated with vehicle or CL 316,243. Representative images show CD31 stain in brown. Mean ± SEM; *p < 0.05, **p < 0.01 vs. vehicle by 1-way ANOVA with Bonferroni post-hoc analysis; n = 8–10.

FIGURE 5

Modified redox signaling after hind limb ischemia was normalized by β₃AR stimulation. (A) Upper panel, representative blot of Nox4 protein expression in hind limb tissue from diabetic mice. Lower panel, quantification. (B) Upper panel, representative blot of Nox2 protein expression in hind limb tissue from diabetic mice. Lower panel, quantification. (C) Upper panel, representative blot of nitrotyrosine expression in hind limb tissue from diabetic mice; Lower panel, quantification of nitrotyrosine expression; In all groups protein expression is shown relative to citrate vehicle non-ischemic limb. Data presented as mean ± SEM. Statistical analysis by 1-way ANOVA with Bonferroni post-hoc analysis. ^# p < 0.05 vs. diabetes vs. citrate; *p < 0.05, **p < 0.01 CL 316,243 vs. vehicle; n = 4.

FIGURE 6

Glutathionylation of eNOS (eNOS-GSS) in ischemic hind limb samples from type 1 (T1) diabetic mice. (A) Representative images of immunoblots (IB) performed on protein fractions following eNOS immunoprecipitation (IP). On the left the expression of GSH, detected at 680 nm is shown and in the middle, the simultaneous expression of eNOS, detected at 800 nm are shown. The right panel shows the merged image of both detection channels. The negative control, -IgG antibody used during IP, is shown only in the top panel. All samples were extracted and run simultaneously. (B) Summary data of eNOS glutathionylation shown as the ratio of glutathionylated eNOS to total eNOS in ischemic hind limb samples. (C) eNOS relative to β-actin expression in total ischemic hindlimb lysate from immunoblot. (D) phosphorylated eNOS (serine 1177) relative to total eNOS in the ischemic hindlimb. Summary data presented as mean ± SEM. Statistical analysis by 1-way ANOVA with Bonferroni post-hoc analysis. ^## p < 0.01 vs. citrate, *p < 0.05 vs. vehicle.

FIGURE 7

β₃AR stimulation improves glucose tolerance and recovery from post-ischemic injury in type 2 diabetes. (A) Schematic of the type 2 diabetes (T2D) protocol. (B) Glucose tolerance tests in a cohort of citrate-buffer control (n = 3–4) and T2D mice treated with vehicle or CL 316,243 (n = 4–5). (C) Area under the curve (AUC) analysis of glucose tolerance results. (D) Ischemic to non-ischemic ratio of perfusion in citrate-buffer treated control (n = 12) and T2D (n = 13) mice measured by laser doppler imaging. Data presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle by 2-way ANOVA.