Interventional- and amputation-stage muscle proteomes in the chronically threatened ischemic limb.

BACKGROUND: Despite improved surgical approaches for chronic limb-threatening ischemia (CLTI), amputation rates remain high and contributing tissue-level factors remain unknown. The purpose of this study was twofold: (1) to identify differences between the healthy adult and CLTI limb muscle proteome, and (2) to identify differences in the limb muscle proteome of CLTI patients prior to surgical intervention or at the time of amputation. METHODS AND RESULTS: Gastrocnemius muscle was collected from non-ischemic controls (n = 19) and either pre-interventional surgery (n = 10) or at amputation outcome (n = 29) CLTI patients. All samples were subjected to isobaric tandem-mass-tag-assisted proteomics. The mitochondrion was the primary classification of downregulated proteins (> 70%) in CLTI limb muscles and paralleled robust functional mitochondrial impairment. Upregulated proteins (> 38%) were largely from the extracellular matrix. Across the two independent sites, 39 proteins were downregulated and 12 upregulated uniformly. Pre-interventional CLTI muscles revealed a robust upregulation of mitochondrial proteins but modest functional impairments in fatty acid oxidation as compared with controls. Comparison of pre-intervention and amputation CLTI limb muscles revealed mitochondrial proteome and functional deficits similar to that between amputation and non-ischemic controls. Interestingly, these observed changes occurred despite 62% of the amputation CLTI patients having undergone a prior surgical intervention. CONCLUSIONS: The CLTI proteome supports failing mitochondria as a phenotype that is unique to amputation outcomes. The signature of pre-intervention CLTI muscle reveals stable mitochondrial protein abundance that is insufficient to uniformly prevent functional impairments. Taken together, these findings support the need for future longitudinal investigations aimed to determine whether mitochondrial failure is causally involved in amputation outcomes from CLTI.

Racial differences in the limb skeletal muscle transcriptional programs of patients with critical limb ischemia.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease (PAD) and is characterized by high rates of morbidity and mortality. As with most severe cardiovascular disease manifestations, Black individuals disproportionately present with CLI. Accordingly, there remains a clear need to better understand the reasons for this discrepancy and to facilitate personalized therapeutic options specific for this population. Gastrocnemius muscle was obtained from White and Black healthy adult volunteers and patients with CLI for whole transcriptome shotgun sequencing (WTSS) and enrichment analysis was performed to identify alterations in specific Reactome pathways. When compared to their race-matched healthy controls, both White and Black patients with CLI demonstrated similar reductions in nuclear and mitochondrial encoded genes and mitochondrial oxygen consumption across multiple substrates, indicating a common bioenergetic paradigm associated with amputation outcomes regardless of race. Direct comparisons between tissues of White and Black patients with CLI revealed hemostasis, extracellular matrix organization, platelet regulation, and vascular wall interactions to be uniquely altered in limb muscles of Black individuals. Among traditional vascular growth factor signaling targets, WTSS revealed only Tie1 to be significantly altered from White levels in Black limb muscle tissues. Quantitative reverse transcription polymerase chain reaction validation of select identified targets verified WTSS directional changes and supports reductions in MMP9 and increases in NUDT4P1 and GRIK2 as unique to limb muscles of Black patients with CLI. This represents a critical first step in better understanding the transcriptional program similarities and differences between Black and White patients in the setting of amputations related to CLI and provides a promising start for therapeutic development in this population.

PFKFB3-mediated glycolysis rescues myopathic outcomes in the ischemic limb.

Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation - critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hind limb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared with the C57BL/6 (BL6) parental strain. AAV-mediated overexpression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing data on muscle from CLI patients revealed a unique deficit in the glucose metabolism Reactome. Muscles from these patients express lower PFKFB3 protein, and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here, we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow-related mitochondrial function is compromised preclinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of the failing CLI patient limb skeletal muscle.

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product-inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.

Extensive skeletal muscle cell mitochondriopathy distinguishes critical limb ischemia patients from claudicants.

The most severe manifestation of peripheral arterial disease (PAD) is critical limb ischemia (CLI). CLI patients suffer high rates of amputation and mortality; accordingly, there remains a clear need both to better understand CLI and to develop more effective treatments. Gastrocnemius muscle was obtained from 32 older (51-84 years) non-PAD controls, 27 claudicating PAD patients (ankle-brachial index [ABI] 0.65 ± 0.21 SD), and 19 CLI patients (ABI 0.35 ± 0.30 SD) for whole transcriptome sequencing and comprehensive mitochondrial phenotyping. Comparable permeabilized myofiber mitochondrial function was paralleled by both similar mitochondrial content and related mRNA expression profiles in non-PAD control and claudicating patient tissues. Tissues from CLI patients, despite being histologically intact and harboring equivalent mitochondrial content, presented a unique bioenergetic signature. This signature was defined by deficits in permeabilized myofiber mitochondrial function and a unique pattern of both nuclear and mitochondrial encoded gene suppression. Moreover, isolated muscle progenitor cells retained both mitochondrial functional deficits and gene suppression observed in the tissue. These findings indicate that muscle tissues from claudicating patients and non-PAD controls were similar in both their bioenergetics profile and mitochondrial phenotypes. In contrast, CLI patient limb skeletal muscles harbor a unique skeletal muscle mitochondriopathy that represents a potentially novel therapeutic site for intervention.

Strain-Dependent Variation in Acute Ischemic Muscle Injury.

Limited efficacy of clinical interventions for peripheral arterial disease necessitates a better understanding of the environmental and genetic determinants of tissue pathology. Existing research has largely ignored the early skeletal muscle injury response during hind limb ischemia (HLI). We compared the hind limb muscle response, after 6 hours of ischemia, in two mouse strains that differ dramatically in their postischemic extended recovery: C57BL/6J and BALB/cJ. Perfusion, measured by laser Doppler and normalized to the control limb, differed only slightly between strains after HLI (<12% across all measures). Similar (<10%) effect sizes in lectin-perfused vessel area and no differences in tissue oxygen saturation measured by reflectance spectroscopy were also found. Muscles from both strains were functionally impaired after HLI, but greater muscle necrosis and loss of dystrophin-positive immunostaining were observed in BALB/cJ muscle compared with C57BL/6J. Muscle cell-specific dystrophin loss and reduced viability were also detected in additional models of ischemia that were independent of residual perfusion differences. Our results indicate that factors other than the completeness of ischemia alone (ie, background genetics) influence the magnitude of acute ischemic muscle injury. These findings may have implications for future development of therapeutic interventions for limb ischemia and for understanding the phasic etiology of chronic and acute ischemic muscle pathophysiology.

Differences in radial expansion force among inferior vena cava filter models support documented perforation rates.

OBJECTIVE: Inferior vena cava (IVC) filters are used in patients at risk for pulmonary embolism who cannot be anticoagulated. Unfortunately, these filters are not without risk, and complications include perforation, migration, and filter fracture. The most prevalent complication is filter perforation of the IVC, with incidence varying among filter models. To our knowledge, the mechanical properties of IVC filters have not been evaluated and are not readily available through the manufacturer. This study sought to determine whether differences in mechanical properties are similar to differences in documented perforation rates. METHODS: The radial expansion forces of Greenfield (Boston Scientific, Marlborough, Mass), Cook Celect (Cook Medical, Bloomington, Ind), and Cook Platinum filters were analyzed with three replicates per group. The intrinsic force exerted by the filter on the measuring device was collected in real time during controlled expansion. Replicates were averaged and significance was determined by calculating analysis of covariance using SAS software (SAS Institute, Cary, NC). RESULTS: Each filter model generated a significantly different radial expansion force (P < .001), and force was distributed at significantly different rates (P < .001) during expansion. The largest radial expansion force at minimal caval diameter was seen in the Cook Platinum filter, followed by the Cook Celect and Greenfield filters. Radial force dispersion during expansion was greatest in the Cook Celect, followed by the Cook Platinum and Greenfield filters. CONCLUSIONS: Differences in radial expansion forces among IVC filter models are consistent with documented perforation rates. Cook Celect IVC filters have a higher incidence of perforation compared with Greenfield filters when they are left in place for >90 days. Evaluation of Cook Celect filters yielded a significantly higher radial expansion force at minimum caval diameter, with greater force dispersion during expansion.

Reporting standards of the Society for Vascular Surgery for endovascular treatment of chronic lower extremity peripheral artery disease: Executive summary.

Recommended reporting standards for lower extremity ischemia were last published by the Society for Vascular Surgery in 1997. Since that time, there has been a proliferation of endovascular therapies for the treatment of chronic peripheral arterial disease. The purpose of this document is to clarify and update these standards, specifically for reports on endovascular treatment. The document is divided into sections: Claudication Reporting, Critical Limb Ischemia Reporting, Preintervention Assessment and Nonanatomic Treatment, Intervention, Outcome Measures - Procedural, Outcome Measures - Disease Specific, and Complications.

Reporting standards of the Society for Vascular Surgery for endovascular treatment of chronic lower extremity peripheral artery disease.

Peripheral arterial disease (PAD) represents a spectrum from asymptomatic stenosis to limb-threatening ischemia. The last decade has seen a tremendous increase in the variety of endovascular devices and techniques to treat occlusive disease. Like many evolving technologies, the literature surrounding therapy for endovascular arterial disease consists of mixed-quality manuscripts without clear standardization. Accordingly, critical evaluation of the reported results may be problematic. As such, providers and their patients make treatment decisions without the full benefit of a comparative effectiveness framework. The purpose of this document is to provide a summary for the reporting of endovascular revascularization techniques in the setting of chronic disease. Much of the work in this document is based on prior publications and standards proposed by the Society for Vascular Surgery. We have also made recommendations based on current literature and have attempted to acknowledge shortcomings and areas for future research. The various sections contain summaries of required reporting standards and should serve as a guide for the design of clinical trials and as reference for journal editors and reviewers when considering scientific work pertaining to endovascular therapy for chronic lower extremity arterial disease. An Appendix is provided with commonly used abbreviations in this document.

Age does not predict need for reintervention in patients with critical limb ischemia.

OBJECTIVE: Conventional wisdom holds that patients with a need for intervention for femoropopliteal occlusive disease at a younger age have more aggressive disease, although there is a paucity of support in the literature. The purpose of this study was to evaluate this assumption. METHODS: A retrospective cohort of patients undergoing endovascular or open revascularization for femoropopliteal occlusive disease for critical limb ischemia during a 4-year period was assembled. Demographic information, comorbidities, disease characteristics, and time to last follow-up, repeat intervention, amputation, or death was recorded. The patients were stratified by age into a young (≤55 years) group, middle (56-77 years) group, and elderly (≥78 years) group. Univariate and multivariate statistical methods were used to evaluate the primary outcome. RESULTS: The study included 124 patients with a mean age of 64.4 ± 0.8 years. Progression to reintervention or amputation occurred in 50% of the patients during the follow-up period, with 18% dying before having an outcome. Kaplan-Meier analysis showed a trend toward significance (P = .06) in time to reintervention, amputation, or death among the three groups, with time to event of 253, 1083, and 504 days for the young, middle, and elderly groups, respectively. However, differences based on age were not significant (P = .57) in Cox regression analysis. CONCLUSIONS: There does not appear to be an association between time to reintervention and patient age.

Impact of psychological factors on objective ambulatory measures in patients with intermittent claudication.

OBJECTIVE: The purpose of this study was to evaluate the difference in objective measures of ambulation and psychosocial factors in patients with intermittent claudication (IC) stratified by type D personality, which incorporates elements of social inhibition and negative affectivity. METHODS: During a 1-year period, routine history and physical examination, ankle-brachial index, and pulse volume recording were performed on IC patients. Questionnaires assessing type D personality and psychosocial factors were also collected. The 6-minute walk test (6MWT) was performed, assessing symptoms and distance walked. Univariate and multivariate methods were used to assess the association between ambulation and type D personality. RESULTS: Seventy-one patients were enrolled (mean age, 62.5 ± 1.1 years; mean ankle-brachial index, 0.55 ± 0.03). Mean distance to symptoms and total distance walked were 83.7 ± 80.1 m and 206.5 ± 126.3 m, respectively. Type D personality was present in 29.6% of the population (n = 21). On 6MWT, 83.1% of all patients developed symptoms, and 57.4% quit because of symptoms. Univariate analysis of objective measures of ambulation demonstrated lower distance to symptoms in the type D group and trends toward lower total distance walked and quitting the 6MWT. Multivariate models showed increased odds of quitting the 6MWT (odds ratio, 7.71; P = .01) and less total distance walked by an average of 33.2 ± 13.3 m (P = .02) for the type D group. CONCLUSIONS: Despite equivalent demographic, medical, and psychosocial factors, the type D group was limited in ambulation, suggesting that type D personality is a strong predictor of disease impact in patients with IC.

Upper extremity thromboembolism in a patient with subclavian steal syndrome.

Subclavian steal is the physiologic process whereby blood flow through a vertebral artery is reversed at the level of the basilar artery as a means of supplying arterial inflow to the ipsilateral subclavian artery. This occurs in the setting of ipsilateral subclavian artery origin occlusion. We describe a case in which a patient with subclavian steal syndrome developed acute upper extremity ischemia secondary to thromboemboli from a chronically occluded ipsilateral subclavian stent (at the origin of the left subclavian artery). He subsequently underwent staged left upper extremity arterial thromboembolectomy followed by definitive revascularization via carotid-subclavian bypass. In addition, subclavian artery ligation proximal to the ipsilateral vertebral artery was performed. The patient's sensory and motor neurologic hand function returned to baseline with restoration of symmetric upper extremity arterial occlusion pressures and pulse volume recordings. A search of the literature revealed that this was the first case report of acute thromboembolic hand ischemia in the setting of subclavian steal.

PACAP regulation of secretion and proliferation of pure populations of gastric ECL cells.

The gastric enterochromaffin-like (ECL) cell plays a major role in the regulation of gastric acid secretion. We have previously described that Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is present on myenteric neurons in the rat and colocalizes with its high-affinity receptor, PAC1, expressed on the surface of gastric ECL cells. The study of ECL cell physiology has been hampered by the inability to isolate and purify ECL cells to homogeneity. Density gradient elutriation alone yields only 65-70% purity of ECL cells. In the present study, we used fluorescence-activated cell sorting (FACS) with a novel fluorescent ligand, Fluor-PACAP-38, for isolating pure ECL cells. FACS was used to isolate ECL cells based on their relatively small size, low density, and ability to bind the fluorescent ligand Fluor-PACAP-38. The sorted cells were unambiguously identified as ECL cells by immunohistochemical analysis using anti-PACAP type-I (PAC1), anti-histidine decarboxylase (HDC), and anti-somatostatin antibodies. Further confocal microscopy demonstrated that Fluor-PACAP-38, a ligand with a higher affinity for PAC1, bound to extracellular receptors of these FACS-purified cells. FACS yielded an average of 2 million ECL cells/4 rat stomachs, and >99% of the sorted cells were positive for PAC1 receptor and HDC expression. The absence of immunohistochemical staining for somatostatin indicated lack of contamination by gastric D cells, which are similar in size and shape to the ECL cells. Internalization of PACAP receptors and a rapid Ca2+ response in purified ECL cells were observed upon PACAP activation, suggesting that these cells are viable and biologically active. These ECL cells demonstrated a dose-dependent stimulation of proliferation in response to PACAP, with a maximum of 30% proliferation at a concentration of 10-7 M. Microarray studies were perfor med to confirm the expression of genes specific for ECL cells. These results demonstrate that rat gastric ECL cells can be isolated to homogeneity by using a combination of density gradient centrifugation, followed by cell sorting using Fluor-PACAP. These techniques now allow microarray studies to be performed in ECL cells to characterize their functional gene expression and will facilitate pharmacological, biochemical, and molecular studies on ECL cell function.

PAC1 and PACAP expression, signaling, and effect on the growth of HCT8, human colonic tumor cells.

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is a heptahelical, G protein-coupled receptor that has been shown to be expressed by non-squamous lung cancer and breast cancer cell lines, and to be coupled to the growth of these tumors. We have previously shown that PACAP and its receptor, PAC1, are expressed in rat colonic tissue. In this study, we used polyclonal antibodies directed against the COOH terminal of PAC1, as well as fluorescently labeled PACAP, Fluor-PACAP, to demonstrate the expression of PAC1 on HCT8 human colonic tumor cells, using FACS analysis and confocal laser scanning microscopy. Similarly, anti-PACAP polyclonal antibodies were used to confirm the expression of PACAP hormone by this cell line. We then investigated the signal transduction properties of PAC1 in these tumor cells. PACAP-38 elevated intracellular cAMP levels in a dose-dependent manner, with a half-maximal (EC(50)) stimulation of approximately 3 nM. In addition, PACAP-38 stimulation caused an increase in cytosolic Ca(2+) concentration [Ca(2+)](i), which was partially inhibited by the PACAP antagonist, PACAP-(6-38). Finally, we studied the potential role of PACAP upon the growth of these tumor cells. We found that PACAP-38, but not VIP, increased the number of viable HCT8 cells, as measured by MTT activity. We also demonstrated that HCT8 cells expressed the Fas receptor (Fas-R/CD95), which was subsequently down-regulated upon activation with PACAP-38, further suggesting a possible role for PACAP in the growth and survival of these tumor cells. These data indicate that HCT8 human colon tumor cells express PAC1 and produce PACAP hormone. Furthermore, PAC1 activation is coupled to adenylate cyclase, increase cytosolic [Ca(2+)](i), and cellular proliferation. Therefore, PACAP is capable of increasing the number of viable cells and regulating Fas-R expression in a human colonic cancer cell line, suggesting that PACAP might play a role in the regulation of colon cancer growth and modulation of T lymphocyte anti-tumoral response via the Fas-R/Fas-L apoptotic pathway.