A 6-minute Limb Function Assessment for Therapeutic Testing in Experimental Peripheral Artery Disease Models.

Background: The translation of promising therapies from pre-clinical models of hindlimb ischemia (HLI) to patients with peripheral artery disease (PAD) has been inadequate. While this failure is multifactorial, primary outcome measures in preclinical HLI models and clinical trials involving patients with PAD are not aligned well. For example, laser Doppler perfusion recovery measured under resting conditions is the most used outcome in HLI studies, whereas clinical trials involving patients with PAD primarily assess walking performance. Here, we sought to develop a 6-min limb function test for preclinical HLI models that assess muscular performance and hemodynamics congruently. Methods: We developed an in situ 6-min limb function test that involves repeated isotonic (shortening) contractions performed against a submaximal load. Continuous measurement of muscle blood flow was performed using laser Doppler flowmetry. Quantification of muscle power, work, and perfusion are obtained across the test. To assess the efficacy of this test, we performed HLI via femoral artery ligation on several mouse strains: C57BL6J, BALBc/J, and MCK-PGC1α (muscle-specific overexpression of PGC1α). Additional experiments were performed using an exercise intervention (voluntary wheel running) following HLI. Results: The 6-min limb function test was successful at detecting differences in limb function of C57BL6/J and BALBc/J mice subjected to HLI with effect sizes superior to laser Doppler perfusion recovery. C57BL6/J mice randomized to exercise therapy following HLI had smaller decline in muscle power, greater hyperemia, and performed more work across the 6-min limb function test compared to non-exercise controls with HLI. Mice with muscle-specific overexpression of PGC1α had no differences in perfusion recovery in resting conditions, but exhibited greater capillary density, increased muscle mass and absolute force levels, and performed more work across the 6-min limb function test compared to their wildtype littermates without the transgene. Conclusion: These results demonstrate the efficacy of the 6-min limb function test to detect differences in the response to HLI across several interventions including where traditional perfusion recovery, capillary density, and muscle strength measures were unable to detect therapeutic differences.

Deletion of the aryl hydrocarbon receptor in endothelial cells improves ischemic angiogenesis in chronic kidney disease.

Chronic kidney disease (CKD) is a strong risk factor for peripheral artery disease (PAD) that is associated with worsened clinical outcomes. CKD leads to the accumulation of tryptophan metabolites that are associated with adverse limb events in PAD and are ligands of the aryl hydrocarbon receptor (AHR), which may regulate ischemic angiogenesis. To test if endothelial cell-specific deletion of the AHR (AHRecKO) alters ischemic angiogenesis and limb function in mice with CKD subjected to femoral artery ligation. Male AHRecKO mice with CKD displayed better limb perfusion recovery and enhanced ischemic angiogenesis compared with wild-type mice with CKD. However, the improved limb perfusion did not result in better muscle performance. In contrast to male mice, deletion of the AHR in female mice with CKD had no impact on perfusion recovery or angiogenesis. With the use of primary endothelial cells from male and female mice, treatment with indoxyl sulfate uncovered sex-dependent differences in AHR activating potential and RNA sequencing revealed wide-ranging sex differences in angiogenic signaling pathways. Endothelium-specific deletion of the AHR improved ischemic angiogenesis in male, but not female, mice with CKD. There are sex-dependent differences in Ahr activating potential within endothelial cells that are independent of sex hormones.NEW & NOTEWORTHY This study provides novel insights into the mechanisms by which chronic kidney disease worsens ischemic limb outcomes in an experimental model of peripheral artery disease. Deletion of the aryl hydrocarbon receptor (AHR) in the endothelium improved ischemic angiogenesis suggesting that AHR inhibition could be a viable therapeutic target; however, this effect was only observed in male mice. Subsequent analysis in primary endothelial cells reveals sex differences in Ahr activating potential independent of sex hormones.

Single-Nuclei RNA-Sequencing of the Gastrocnemius Muscle in Peripheral Artery Disease.

BACKGROUND: Lower extremity peripheral artery disease (PAD) is a growing epidemic with limited effective treatment options. Here, we provide a single-nuclei atlas of PAD limb muscle to facilitate a better understanding of the composition of cells and transcriptional differences that comprise the diseased limb muscle. METHODS: We obtained gastrocnemius muscle specimens from 20 patients with PAD and 12 non-PAD controls. Nuclei were isolated and single-nuclei RNA-sequencing was performed. The composition of nuclei was characterized by iterative clustering via principal component analysis, differential expression analysis, and the use of known marker genes. Bioinformatics analysis was performed to determine differences in gene expression between PAD and non-PAD nuclei, as well as subsequent analysis of intercellular signaling networks. Additional histological analyses of muscle specimens accompany the single-nuclei RNA-sequencing atlas. RESULTS: Single-nuclei RNA-sequencing analysis indicated a fiber type shift with patients with PAD having fewer type I (slow/oxidative) and more type II (fast/glycolytic) myonuclei compared with non-PAD, which was confirmed using immunostaining of muscle specimens. Myonuclei from PAD displayed global upregulation of genes involved in stress response, autophagy, hypoxia, and atrophy. Subclustering of myonuclei also identified populations that were unique to PAD muscle characterized by metabolic dysregulation. PAD muscles also displayed unique transcriptional profiles and increased diversity of transcriptomes in muscle stem cells, regenerating myonuclei, and fibro-adipogenic progenitor cells. Analysis of intercellular communication networks revealed fibro-adipogenic progenitors as a major signaling hub in PAD muscle, as well as deficiencies in angiogenic and bone morphogenetic protein signaling which may contribute to poor limb function in PAD. CONCLUSIONS: This reference single-nuclei RNA-sequencing atlas provides a comprehensive analysis of the cell composition, transcriptional signature, and intercellular communication pathways that are altered in the PAD condition.

Activation of the Aryl Hydrocarbon Receptor in Endothelial Cells Impairs Ischemic Angiogenesis in Chronic Kidney Disease.

Rationale: Chronic kidney disease (CKD) is a strong risk factor for peripheral artery disease (PAD) that is associated with worsened clinical outcomes. CKD leads to accumulation of tryptophan metabolites that associate with adverse limb events in PAD and are ligands of the aryl hydrocarbon receptor (AHR) which may regulate ischemic angiogenesis. Objectives: To test if endothelial cell-specific deletion of the AHR (AHRecKO) alters ischemic angiogenesis and limb function in mice with CKD subjected to femoral artery ligation. Findings: Male AHRecKO mice with CKD displayed better limb perfusion recovery and enhanced ischemic angiogenesis compared to wildtype mice with CKD. However, the improved limb perfusion did not result in better muscle performance. In contrast to male mice, deletion of the AHR in female mice with CKD had no impact on perfusion recovery or angiogenesis. Using primary endothelial cells from male and female mice, treatment with indoxyl sulfate uncovered sex-dependent differences in AHR activating potential and RNA sequencing revealed wide ranging sex-differences in angiogenic signaling pathways. Conclusion: Endothelium-specific deletion of the AHR improved ischemic angiogenesis in male, but not female, mice with CKD. There are sex-dependent differences in Ahr activating potential within endothelial cells that are independent of sex hormones.

Chronic activation of the aryl hydrocarbon receptor in muscle exacerbates ischemic pathology in chronic kidney disease.

Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the cellular and physiological mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic toxins, many of which are ligands for the aryl hydrocarbon receptor (AHR), are associated with adverse limb outcomes in PAD. We hypothesized that chronic AHR activation, driven by the accumulation of tryptophan-derived uremic metabolites, may mediate the myopathic condition in the presence of CKD and PAD. Both PAD patients with CKD and mice with CKD subjected to femoral artery ligation (FAL) displayed significantly higher mRNA expression of classical AHR-dependent genes ( Cyp1a1 , Cyp1b1 , and Aldh3a1 ) when compared to either muscle from the PAD condition with normal renal function ( P <0.05 for all three genes) or non-ischemic controls. Skeletal-muscle-specific AHR deletion in mice (AHR mKO ) significantly improved limb muscle perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and contractile function, as well as enhanced mitochondrial oxidative phosphorylation and respiratory capacity in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. These findings establish chronic AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in PAD. Further, the totality of the results provide support for testing of clinical interventions that diminish AHR signaling in these conditions.

Activation of the Aryl Hydrocarbon Receptor in Muscle Exacerbates Ischemic Pathology in Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic solutes, which are ligands for AHR (aryl hydrocarbon receptor), are associated with limb amputation in PAD. Herein, we examined the role of AHR activation in the myopathy of PAD and CKD. METHODS: AHR-related gene expression was evaluated in skeletal muscle obtained from mice and human PAD patients with and without CKD. AHRmKO (skeletal muscle-specific AHR knockout) mice with and without CKD were subjected to femoral artery ligation, and a battery of assessments were performed to evaluate vascular, muscle, and mitochondrial health. Single-nuclei RNA sequencing was performed to explore intercellular communication. Expression of the constitutively active AHR was used to isolate the role of AHR in mice without CKD. RESULTS: PAD patients and mice with CKD displayed significantly higher mRNA expression of classical AHR-dependent genes (Cyp1a1, Cyp1b1, and Aldh3a1) when compared with either muscle from the PAD condition with normal renal function (P<0.05 for all 3 genes) or nonischemic controls. AHRmKO significantly improved limb perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and strength, as well as enhanced mitochondrial function in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. CONCLUSIONS: These findings establish AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in CKD. Further, the totality of the results provides support for testing of clinical interventions that diminish AHR signaling in these conditions.