Mouse Model Of Peripheral Arterial Disease Research Articles

Comparison effect of high-intensity interval training and moderate intensity continuous training on vascular function in a mouse model of lower extremity peripheral artery disease

Introduction
 Lower extremity peripheral artery disease (PAD), due to narrowing of the arteries supplying the legs, represents a major cause of physical disability. Exercise training is an important part of PAD treatment, improving functional capacity. Recent research suggests that exercise training-induced endothelial function improvement is involved in the benefits of exercise in PAD (Lee et al., 2023); however, the underlying molecular mechanisms remain unknown. In addition, the potential vascular benefits of high intensity training in PAD remain unexplored. Therefore, the aim of the study was to compare the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on endurance capacity, vascular endothelial function and expression of genes related to nitric oxide (NO) bioavailability in a mouse model of PAD.
 Methods
 PAD disease model was generated in male C57BL/6 mice by ligation of the right iliac artery. Mice with PAD were divided into 3 groups: sedentary control group (SED), MICT (40 min running at 70% of maximal aerobic speed) and HIIT (8 intervals of 2.5 minutes running at 90% of maximal aerobic speed combined with 2.5 minutes of running at 50% of maximal aerobic speed). Mice were exercised three times per week for 8 weeks. Maximal running time was determined by an incremental test until exhaustion. Vascular reactivity was assessed by measuring vasoconstrictor responses to phenylephrine as well as endothelium-dependent and -independent vasodilator responses to acetylcholine and DEA-NONOate, respectively, in isolated aortic rings. Gene expression of antioxidant enzymes (catalase and superoxide dismutase isoforms), prooxidant NADPH oxidase subunits (p22phox and p47phox), enzyme involved in NO production (endothelial NO synthase), vascular smooth muscle cell relaxation (soluble guanylate cyclase) and receptors involved in vascular tone regulation (a1 and b2 adrenergic receptors) was determined in aortic tissue by qPCR.
 Results
 Maximal running time was improved in both MICT and HIIT as compared to SED at the end of the study, but with no significant difference between the two exercise groups. Phenylephrine-induced vasoconstriction as well as vasodilator responses to acetylcholine and DEA-NONOate were significantly improved in MICT compared to SED while no significant differences were reported between HIIT and SED. There was no significant difference in gene expression among the three groups.
 Discussion/Conclusion
 MICT and HIIT were equally effective at improving endurance capacity in mice with PAD. However, only MICT improved vascular reactivity.
 References
 Lee, J., Zarezadehmehrizi, A., LaVoy, E., Markofski, M., & Park, Y. (2023). Exercise training improves brachial artery endothelial function, but does not alter inflammatory biomarkers in patients with peripheral artery disease: A systematic review and meta-analysis. Journal of Cardiovascular Translational Research. Advance online publication. https://doi.org/10.1007/s12265-023-10451-0

Abstract 17363: Loss of Skeletal Muscle Bmal1 Impairs Limb Perfusion and Muscle Regeneration in a Mouse Model of Peripheral Arterial Disease

Introduction: Peripheral arterial disease (PAD) is a growing pathologic condition worldwide and leads to severe myopathy. Traditional risk factors for PAD, including diabetes, smoking, and advanced age, are associated with disrupted circadian clock function. However, the contribution of circadian disruption to PAD remains poorly understood. Hypothesis: This study aims to investigate the role of skeletal muscle clock in the severity of PAD. Methods: We performed femoral artery ligation (FAL) surgery in male adult-life inducible skeletal muscle-specific Bmal1 knockout ( Bmal1 musc ) (n = 6) and control mice (n = 5). We measured limb perfusion with laser doppler imaging weekly and assessed post-surgical angiogenesis with CD31/αSMA co-immunostaining and muscle recovery with hematoxylin and eosin (H&E) staining and Laminin immunohistochemistry at 30 days post-surgery. We also performed RNA-sequencing, qPCR, and Western blotting assays to characterize the differentially expressed genes in both ligated and non-ligated hindlimb muscles. Results: Adult-life deletion of skeletal muscle Bmal1 reduced reperfusion recovery, with an average rate of only 50% by 22 days post-surgery in Bmal1 musc mice compared to approximately 80% in control mice. HIF1α target genes including the pro-angiogenic factor Vegfα were significantly downregulated in Bmal1 musc mice. Conversely, genes enriched in the anti-angiogenic TGF-β signaling pathways were up-regulated in Bmal1 musc mice. Myostatin, a myokine that inhibits muscle growth, was downregulated in Bmal1 musc muscles. H&E staining and Laminin immunohistochemistry revealed significantly less but enlarged newly formed myofibers in the tibialis anterior muscle in Bmal1 musc mice. Conclusions: Skeletal muscle BMAL1 plays a critical role in determining the severity of ischemic muscle injury in a mouse model of PAD. Circadian dysfunction impairs hindlimb ischemia reperfusion, decreases the number of newly formed myofibers, and promotes the formation of enlarged regenerating myofibers, which is however previously reported to be associated with compromised muscle force production. Restore muscle circadian function could be a promising strategy to ameliorate myopathic severity in PAD patients.

Contrast-Enhanced Ultrasound Reveals Partial Perfusion Recovery After Hindlimb Ischemia as Opposed to Full Recovery by Laser Doppler Perfusion Imaging.

Mouse models are critical in developing new therapeutic approaches to treat peripheral arterial disease (PAD). Despite decades of research and numerous clinical trials, the efficacy of available therapies is limited. This may suggest shortcomings in our current animal models and/or methods of assessment. We evaluated perfusion measurement methods in a mouse model of PAD by comparing laser Doppler perfusion imaging (LDPI, the most common technique), contrast-enhanced ultrasound (CEUS, an emerging technique) and fluorescent microspheres (conventional standard). Mice undergoing a femoral artery ligation were assessed by LDPI and CEUS at baseline and 1, 4, 7, 14, 28, 60, 90 and 150 d post-surgery to evaluate perfusion recovery in the ischemic hindlimb. Fourteen days after surgery, additional mice were measured with fluorescent microspheres, LDPI, and CEUS. LDPI and CEUS resulted in broadly similar trends of perfusion recovery until 7 d post-surgery. However, by day 14, LDPI indicated full recovery of perfusion, whereas CEUS indicated ∼50% recovery, which failed to improve even after 5 mo. In agreement with the CEUS results, fluorescent microspheres at day 14 post-surgery confirmed that perfusion recovery was incomplete. Histopathology and photoacoustic microscopy provided further evidence of sustained vascular abnormalities.

Abstract 11323: Role of Mir-6236 on Skeletal Muscle Angiogenesis

Peripheral arterial disease (PAD) affects more than 200 million people worldwide and is characterized by impaired blood flow to the lower extremities. There are no medical treatments available and in severe cases, PAD may lead to critical limb ischemia requiring amputation. The pathophysiology of PAD is characterized by marked changes in gene expression and reduced angiogenesis. Although many studies have investigated the molecular mechanisms involved in PAD, less is known about the role of microRNAs (miRNA) in the pathogenesis of the disease. MiRNAs are important regulators of gene expression that bind to specific target mRNA and repress their expression. Using a mouse model of PAD, we investigated miRNAs induced by ischemia using RNAseq analysis of ischemic and non-ischemic hind limb tissue. Our data showed 77 miRNAs were upregulated and 40 miRNAs were downregulated in ischemic mouse hind limbs versus non-ischemic controls. Among these miRNAs, miR-6236 was significantly upregulated suggesting a possible role in post ischemic adaptation. We validated miR-6236 expression by qPCR (Ischemic 2.04±0.39 vs Non-Ischemic 1.0±0.11; n=5-6; p<0.05). In vitro, using primary mouse microvascular endothelial cells (MVECs), ischemia induced miR-6236 expression in a time dependent manner up to 72 hrs. At the same time, ischemia caused decrease in proliferation (Ischemia (Isch) 0.12±0.03 vs Normoxia (Norm) 0.23±0.02; n=4-6; p<0.05), migration (Isch 46.7±0.52 vs Norm 72.02±2.74; n=6; p<0.05) and tube formation (Isch 31.5±3.58 vs Norm 82.12±6.5; n=8; p<0.05), and increased apoptosis (Isch 0.94±0.05 vs Norm 0.59±0.02; n=4-5; p<0.05). Treatment of mouse MVECs with miR-6236 inhibitor (miRINH, 100 nM) prior to ischemia decreased miR-6236 expression by 50% and improved proliferation (miRINH + Isch 0.21±0.01 vs Isch 0.12±0.03; n= 4; p<0.05), migration (miRINH + Isch 63.71±2.08 vs Isch 46.7±0.52; n=6; p<0.05), tube formation (miRINH + Isch 50±5.92 vs Isch 31.5±3.58; n=8; p<0.05) and decreased ischemia-induced apoptosis (miRINH + Isch 0.64±0.04 vs Isch 0.94±0.05; n=4-5; p<0.05). We conclude that miR-6236 serves as a critical regulator of endothelial cell function in ischemic skeletal muscle and can be a promising target for therapeutic skeletal muscle angiogenesis.

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.

Gold Nanorods as Photoacoustic Nanoprobes to Detect Proinflammatory Macrophages and Inflammation

Key hallmarks of inflammation include proinflammatory (M1) macrophages and production of reactive oxygen species (ROS). Therefore, we developed a ROS-sensitive, photoacoustic (PA) nanoprobe capable of identifying M1 macrophages in vitro. The nanoprobe is a silica-coated gold nanorod (AuNR), which provides a stable PA signal, combined with an adsorbed polyelectrolyte-dye (poly-d-lysine (PDL)-IR775c) coating that exhibits ROS sensitivity. By monitoring the ratio between these two PA intensities, we can determine if ROS is present. Upon loading, then stimulating, macrophages toward an M1 phenotype, we observe a significant decrease in the ratio of dye/AuNR intensities relative to a nonstimulated control. Our nanoprobe is also applied to a mouse model of peripheral artery disease (PAD), where we demonstrate an ability to identify early signs of muscle inflammation before symptoms or histologic evidence. Taken together, our nanoprobe can be a powerful tool for monitoring macrophage phenotype and inflammation in vivo.

Near-Infrared IIb Fluorescence Imaging of Vascular Regeneration with Dynamic Tissue Perfusion Measurement and High Spatial Resolution.

Real-time optical imaging is a promising approach for visualizing in vivo hemodynamics and vascular structure in mice with experimentally induced peripheral arterial disease (PAD). We report the application of a novel fluorescence-based all-optical imaging approach in the near-infrared IIb (NIR-IIb, 1500-1700 nm emission) window, for imaging hindlimb microvasculature and blood perfusion in a mouse model of PAD. In phantom studies, lead sulfide/cadmium sulfide (PbS/CdS) quantum dots showed better retention of image clarity, in comparison with single-walled nanotube (SWNT) NIR-IIa (1000-1400nm) dye, at varying depths of penetration. When systemically injected to mice, PbS/CdS demonstrated improved clarity of the vasculature, compared to SWNTs, as well as higher spatial resolution than in vivo microscopic computed tomography. In a mouse model of PAD, NIR-IIb imaging of the ischemic hindlimb vasculature showed significant improvement in blood perfusion over the course of 10 days (P<0.05), as well as a significant increase in microvascular density over the first 7 days after induction of PAD. In conclusion, NIR-IIb imaging of PbS/CdS vascular contrast agent is a useful multi-functional imaging approach for high spatial resolution imaging of the microvasculature and quantification of blood perfusion recovery.

ImmunoPET of CD146 in a Murine Hindlimb Ischemia Model.

Peripheral arterial disease (PAD) consists of a persistent obstruction of lower-extremity arteries further from the aortic bifurcation attributable to atherosclerosis. PAD is correlated with an elevated risk of morbidity and mortality as well as of deterioration of the quality of life with claudication and chronic leg ischemia being the most frequent complications. Therapeutic angiogenesis is a promising therapeutic strategy that aims to restore the blood flow to the ischemic limb. In this context, assessing the efficacy of pro-angiogenic treatment using a reliable noninvasive imaging technique would greatly benefit the implementation of this therapeutic approach. Herein, we describe the angiogenesis and perfusion recovery characteristics of a mouse model of PAD via in vivo positron emission tomography (PET) imaging of CD146 expression. For that, ischemia was generated by ligation and excision of the right femoral artery of Balb/C mice and confirmed through laser Doppler imaging. The angiogenic process, induced by ischemia, was noninvasively monitored and quantified through PET imaging of CD146 expression in the injured leg using a 64Cu-labeled anti-CD146 monoclonal antibody, 64Cu-NOTA-YY146, at post-operative days 3, 10, and 17. The CD146-specific character of 64Cu-NOTA-YY146 was verified via a blocking study performed in another cohort at day 10 after surgery. Tracer uptake was correlated with in situ CD146 expression by histological analysis. PET scan results indicated that 64Cu-NOTA-YY146 uptake in the injured leg was significantly higher, with the highest uptake with a value of 14.1 ± 2.0 %ID/g at post-operative day 3, compared to the normal contralateral hindlimb, at all time points (maximum uptake of 2.2 ± 0.2 %ID/g). The pre-injection of a blocking dose resulted in a significantly lower tracer uptake in the ischemic hindlimb on day 10 after surgery, confirming tracer specificity. CD146/CD31 immunofluorescent co-staining showed an excellent correlation between the high uptake of the tracer with in situ CD146 expression levels and a marked co-localization of CD146 and CD31 signals. In conclusion, persistent and CD146-specific tracer accumulation in the ischemic hindlimb was observed, confirming the feasibility of 64Cu-NOTA-YY146 to be used as an imaging agent to monitor the progression of angiogenesis and recovery in future PAD research.

Impact of heat therapy on skeletal muscle structure and function in a mouse model of peripheral arterial disease

IntroductionAlterations in skeletal muscle morphology and function, including increased connective tissue infiltration and reduced strength, contribute to exercise intolerance in patients with symptomatic peripheral arterial disease (PAD). We tested the hypothesis that repeated exposure to heat therapy (HT) would reverse skeletal muscle abnormalities in a mouse model of PAD.MethodsMale 42‐week‐old C57Bl/6 mice underwent bilateral ligation of the femoral artery to induce hindlimb ischemia. After two weeks of recovery, the animals were randomly assigned to receive HT or a control intervention. Heat therapy was applied by placing the animal in a flat bottom restrainer and immersing the lower half of the body in a glass container filled with water at 37°C (n=12), 39°C (n=12), or 41°C (n=12) for 30 min daily over 3 consecutive weeks. Animals assigned to the control group (n=12) were also restrained, but were placed in an empty container. Rectal temperature was measured before and during exposure to a single session of HT or the control intervention (n=2/group). Forty‐eight hours following the last treatment session, the animals were anesthetized and the soleus and extensor digitorium longus (EDL) muscles were harvested for the assessment of contractile function in vitro and histological determination of fiber morphology and collagen infiltration.ResultsRectal temperature increased rapidly during exposure to HT and reached a steady state within approximately 10 min (37ºC: 37.1±0.1°C, 39ºC: 38.7±0.2°C, 41ºC: 40.1±0.1°C), while in the control group rectal temperature remained at baseline levels throughout the session (34.9±1.1°C). Muscle weight relative to body weight was significantly higher in animals exposed to HT at 39ºC as compared to the control group in both the soleus (Control: 0.36±0.01 mg/g vs. 39HT: 0.41±0.01 mg/g, p=0.023) and EDL muscles (Control: 0.43±0.01 mg/g vs. 39HT: 0.48±0.01 mg/g, p=0.029). Maximal absolute force of the soleus tended to be higher in animals treated with HT at 37ºC (p=0.072) and at 39ºC (p=0.108) when compared to the control group (Control: 274.3±7.7 mN, 37ºC: 303.0±7.8 mN, 39 ºC: 301.1±13.5 mN). In the EDL muscle, collagen content was lower in the group treated with HT at 37ºC (Control: 5.1±1.3% vs. 37ºC: 2.9±0.8%, p=0.028). Similarly, there was a tendency for lower collagen infiltration in the soleus muscle in the group treated with HT at 39ºC when compared to the control group (Control: 5.7±0.8% vs. 39ºC: 4.6±1.4%, p=0.101). There were no differences between groups in specific muscle force, number of fibers with central nucleation and fiber‐cross sectional area in both the EDL and soleus muscles.ConclusionThese findings suggest that repeated exposure to HT at 37°C and 39°C, but not at 41°C, ameliorates skeletal muscle abnormalities in a mouse model of PAD.Support or Funding InformationFunding: Ralph W. and Grace M. Showalter Research Trust AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The angiogenic properties of human adipose-derived stem cells (HASCs) are modulated by the High mobility group box protein 1 (HMGB1)

Peripheral arterial disease (PAD), is a major health problem. Many studies have been focused on the possibilities of treatment offered by vascular regeneration. Human adipose-derived stem cells (HASCs), multipotent CD34+ stem cells found in the stromal-vascular fraction of adipose tissues, which are capable to differentiate into multiple mesenchymal cell types. The High mobility group box 1 protein (HMGB1) is a nuclear protein involved in angiogenesis.The aim of the study was to define the role of HMGB1 in cell therapy with HASCs, in an animal model of PAD.We induced unilateral ischemia in mice and we treated them with HASCs, with the specific HMGB1-inihibitor BoxA, with HMGB1 protein, and with the specific VEGF inhibitor sFlt1, alternately or concurrently. We measured the blood flow recovery in all mice. Immunohistochemical and ELISA analyses was performed to evaluate the number of vessels and the VEGF tissue content.None auto-amputation occurred and there have been no rejection reactions to the administration of HASCs. Animals co-treated with HASCs and HMGB1 protein had an improved blood flow recovery, compared to HASCs-treated mice. The post-ischemic angiogenesis was reduced when the HMGB1 pathway was blocked or when the VEGF activity was inhibited, in mice co-treated with HASCs and HMGB1.In conclusion, the HASCs treatment can be used in a mouse model of PAD to induce post-ischemic angiogenesis, modulating angiogenesis by HMGB1. This effect is mediated by VEGF activity. Although further data are needed, these findings shed light on possible new cell treatments for patients with PAD.

Endothelial Foxo1 and Foxo3a Synergistically Repress Skeletal Muscle Vascular Growth and Blood Flow Recovery in a Mouse Model of Hind Limb Ischemia.

Neovascularization in peripheral artery disease (PAD) is impaired despite upregulation of factors that promote blood vessel growth which suggest a concomitant increase of anti‐angiogenic repressors. We previously showed that depletion of FoxO proteins (FoxO1/3a/4) induced angiogenesis and enhanced blood flow recovery in a mouse model of PAD. The purpose of this study is to independently assess the roles of endothelial FoxO1 and synergistic actions of FoxO 1 &amp; 3a in a mouse model of PAD. Mice with tamoxifen‐inducible endothelial specific deletions of FoxO1 (PDGFBCreERT2+;FoxO1L/L, FoxO1Δ) and FoxO1/3a (PDGFBCreERT2+;FoxO1/3aL/L, FoxO1/3Δ), or control (PDGFBCreERT2‐, FoxO1L/L) mice, underwent unilateral ligation of the common femoral artery (at ~8–9 weeks of age) to induce a substantial (~80%) reduction of blood flow in the ligated (LIG) compared to the contralateral (CON) leg. Blood flow recovery was analyzed by laser doppler imaging at baseline, then on days 4, 7, 10 and 14 post‐ligation. Muscle histology was conducted after 14 days. FoxO1/3Δ blood flow (LIG/CON leg perfusion ratio) was recovered to basal levels (1.10 ±0.07; n=6) and significantly greater than FoxO1Δ (0.73 ±0.05; P&lt;0.01, n=5) and FoxO1L/L (0.65 ±0.03; P&lt;0.001, n=5) mice 14 days post‐ligation. Neither FoxO1 nor FoxO1/3 deletion altered capillary density (CD) in the control leg. In contrast, CON leg capillary‐to‐fibre ratio (C: F) in FoxO1/3Δ mice (1.02 ±0.04) was significantly lower than FoxO1L/L (1.48 ±0.08) and FoxO1Δ (1.44 ±0.09) mice (P &lt;0.05). Ischemic muscle was characterized by marked cellular infiltration, deposition of fibrous and adipose tissue and myofibre atrophy. Capillary density was significantly increased in ischemic muscles from FoxO1Δ (CON: 746 ±40.15 vs LIG: 1218 ±187.4 capillaries/mm2; P =0.03) and FoxO1/3aΔ (CON: 743.2 ±39.35 vs LIG: 1661 ±158.5 capillaries/mm2; P =0.005) mice. C: F was increased ~25% in ischemic muscle from FoxO1/3Δ mice (1.28 ±0.05; P &lt;0.05) but was unaltered in FoxO1L/L (1.46 ±0.19) or FoxO1Δ mice (1.55 ±0.22). Our data suggest that the combined effects of FoxO1 &amp; 3a repress vascular growth and blood flow recovery in ischemic muscles more than the independent effects of FoxO1. Funded by CIHR.

Abstract 510: Perivascular-Cell Derived Oct4 is Essential for Angiogenesis Following Hindlimb Ischemia

Ischemic vascular diseases such as coronary, carotid, and peripheral artery disease (PAD) require vascular growth and remodeling in order to restore blood flow to ischemic tissue. Human therapeutic studies aimed at promoting neovascularization have been largely unsuccessful in part because these approaches have focused on stimulating endothelial cell (EC) growth without coordinate investment of smooth muscle cells and pericytes (SMC-P) needed for functional blood vessels that are stable and able to regulate perfusion. Thus, there is a critical need to identify mechanisms that mediate perivascular cell coverage during vascular network remodeling, a process that requires perivascular cell detachment, migration, and investment of endothelial tubes. Previous work from the Owens lab demonstrated that the stem cell pluripotency gene Oct4, previously thought to be permanently epigenetically silenced in all somatic cells, is reactivated in SMC within atherosclerotic lesions. SMC specific KO of Oct4 results in marked reductions in the number of SMC within lesions due to failure of cells to migrate out of the media. That is, Oct4 reactivation appears to be critical for SMC migration, at least in the context of atherosclerosis. Therefore, the present study tested the hypothesis that SMC-P reactivation of Oct4 is essential for neovascularization following hindlimb ischemia (HLI), a mouse model of PAD. To test this hypothesis, we used a unique mouse line enabling SMC-P specific tamoxifen-inducible lineage tracing (termed Myh11 eYFP), allowing their tracking following phenotypic switching and loss of identifying genes, combined with SMC-P specific knockout of Oct4. We subjected both Oct4WT and Oct4KO Myh11 eYFP mice to HLI and monitored perfusion recovery via Laser Doppler at days 0, 3, 7, 14, and 21 post-HLI. We found that SMC-P specific KO of Oct4 results in significantly impaired perfusion recovery at days 14 and 21 post-HLI. We harvested hindlimb muscle at day 21 post-HLI and observed that Oct4 KO mice have significantly impaired angiogenesis, but intact arteriogenesis, compared to WT controls. Future studies will investigate Oct4 downstream targets necessary for angiogenesis following HLI.

Ultrasound-microbubble targeted delivery of ICAM-1 in mouse model of peripheral arterial disease

Peripheral arterial disease (PAD) affects 12 million people in the United States. Unfortunately, current treatments are highly invasive and have limited efficacy. Ultrasound (US) in conjunction with microbubbles (MB) has recently been explored as a non-invasive strategy for the delivery of intravascularly circulating therapeutic agents. US mediated MB oscillation can lead to non-damaging, reversible and localized vascular permeabilization resulting in substantial increases of nanoparticle (NP) concentrations in US-treated tissue. Furthermore, by coating NPs with a brush layer of polyethylene glycol (PEG), NPs have long circulating half-lives and limited toxicity. This study investigates the ability of US to deliver ICAM-1-bearing PEG/polyethylenimine (PEG/PEI) NPs to the vascular endothelium in a mouse model of PAD. ICAM-1, an adhesion molecule, expressed post-occlusion by the endothelium of collateral arteries has previously been shown to aid in blood flow restoration. Overexpression of ICAM-1 in this model aims to re-establish pre-occlusion blood flow non-invasively.

An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease.

Peripheral artery disease (PAD) generates tissue ischemia through arterial occlusions and insufficient collateral vessel formation. Vascular insufficiency in PAD occurs despite higher circulating levels of vascular endothelial growth factor A (VEGF-A),1,2 a key regulator of angiogenesis. Here, we show that clinical PAD is associated with elevated anti-angiogenic VEGF-A splice isoform (VEGF-A165b), and a corresponding reduction of the pro-angiogenic VEGF-A165a isoform. In a murine model of PAD, VEGF-A165b was upregulated by conditions associated with impaired limb revascularization, including leptin-deficiency, diet-induced obesity, genetic ablation of the secreted frizzled-related protein 5 (Sfrp5) adipokine and transgenic overexpression of Wnt5a in myeloid cells. In PAD models, delivery of VEGF-A165b inhibited revascularization of ischemic hind limbs, whereas treatment with an isoform-specific neutralizing antibody reversed the impaired revascularization phenotype caused by metabolic dysfunction or perturbations in the Wnt5a/Sfrp5 regulatory system. These results indicate that inflammation driven expression of the anti-angiogenic VEGF-A isoform can contribute to impaired collateralization in ischemic cardiovascular disease.

Uncoupling angiogenesis and inflammation in peripheral artery disease with therapeutic peptide-loaded microgels

Peripheral artery disease (PAD) is characterized by vessel occlusion and ischemia in the limbs. Treatment for PAD with surgical interventions has been showing limited success. Moreover, recent clinical trials with treatment of angiogenic growth factors proved ineffective as increased angiogenesis triggered severe inflammation in a proportionally coupled fashion. Hence, the overarching goal of this research was to address this issue by developing a biomaterial system that enables controlled, dual delivery of pro-angiogenic C16 and anti-inflammatory Ac-SDKP peptides in a minimally-invasive way. To achieve the goal, a peptide-loaded injectable microgel system was developed and tested in a mouse model of PAD. When delivered through multiple, low volume injections, the combination of C16 and Ac-SDKP peptides promoted angiogenesis, muscle regeneration, and perfusion recovery, while minimizing detrimental inflammation. Additionally, this peptide combination regulated inflammatory TNF-α pathways independently of MMP-9 mediated pathways of angiogenesis in vitro, suggesting a potential mechanism by which angiogenic and inflammatory responses can be uncoupled in the context of PAD. This study demonstrates a translatable potential of the dual peptide-loaded injectable microgel system for PAD treatment.

Effects of N-acetylcysteine on skeletal muscle structure and function in a mouse model of peripheral arterial insufficiency

Abnormalities in skeletal muscle structure and function are important contributors to exercise intolerance and functional decline in peripheral arterial disease. In this study, we tested the hypothesis that administration of N-acetylcysteine (NAC) would improve fatigue resistance and ameliorate the histopathological changes in skeletal muscle in a mouse model of peripheral arterial disease. We also anticipated that NAC treatment would lower the levels of biomarkers of oxidative damage in the ischemic muscle. Male Balb/c mice were subjected to bilateral ligation of the femoral artery and, after 2 weeks of recovery, received daily intraperitoneal injections of either NAC (150 mg/kg) or saline for 15 days. At the end of the treatment, the extensor digitorium longus (EDL) and soleus muscles were excised for assessment of contractile function in vitro and histological analysis. Free malondialdehyde and protein carbonyl levels were measured in the gastrocnemius muscle. In the soleus muscle, force after 10 minutes of submaximal tetanic stimulation (60 Hz, 300 ms trains, 0.3 trains/s) was higher (P < .05) in NAC-treated animals (45% ± 3% of the initial value; n = 7) when compared with controls (30.3% ± 3%; n = 8). No differences were found in fatigue development between groups in the EDL muscle (ligated NAC, 35.7% ± 1.9%; ligated saline, 37.5% ± 1.1%). In addition, there was a tendency for lower levels of connective tissue deposition in the soleus of animals treated with NAC (n = 6) when compared with those that received only saline (n = 9) (ligated NAC, 16% ± 2% vs ligated saline, 24% ± 2%; P = .057). No differences were found in lipid peroxidation or protein carbonyl levels between ligated saline and ligated NAC groups. Taken together, these results indicate that treatment with NAC improves fatigue resistance in the soleus but not the EDL muscle in a model of peripheral arterial insufficiency. Despite the increasing burden of peripheral arterial disease (PAD) and its detrimental consequences on the quality of life of the patients, few pharmacological therapies have shown to evoke meaningful effects on functional performance in these individuals. N-acetylcysteine is approved for clinical use, has minimal side effects and most important, has shown to consistently improve exercise performance in animals and humans. In this study, we showed, for the first time, that treatment with this drug at a dose amenable for clinical application evoked marked effects on fatigue resistance in the soleus muscle in a mouse model of PAD. These encouraging findings set the stage for translational studies to determine the acute and long-term impact of this drug on walking capacity in patients with PAD.

Unacylated Ghrelin Promotes Skeletal Muscle Regeneration Following Hindlimb Ischemia via SOD‐2–Mediated miR‐221/222 Expression

BackgroundSurgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated‐ghrelin (UnAG) to reduce ischemia‐induced tissue damage in a mouse model of peripheral artery disease.Methods and ResultsWe show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen‐actived protein kinase signaling UnAG protected against reactive oxygen species–induced cell injuries by inducing the expression of superoxide dismutase‐2 (SOD‐2) in satellite cells. This led to a reduced number of infiltrating CD68+ cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR‐221/222, previously linked to muscle regeneration processes, was up‐regulated and negatively correlated with p57Kip2 expression in UnAG‐treated mice. UnAG, unlike AG, promoted cell‐cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG‐induced p38/mitogen‐actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD‐2–depleted SCs. By siRNA technology, we also demonstrated that SOD‐2 is the antioxidant enzyme involved in the control of miR‐221/222–driven posttranscriptional p57Kip2 regulation. Loss‐of‐function experiments targeting miR‐221/222 and local pre–miR‐221/222 injection in vivo confirmed a role for miR‐221/222 in driving skeletal muscle regeneration after ischemia.ConclusionsThese results indicate that UnAG‐induced skeletal muscle regeneration after ischemia depends on SOD‐2–induced miR‐221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species–mediated skeletal muscle damage.