Abstract 17327: Ischemic Skeletal Muscle From Patients With Peripheral Arterial Disease Displays Marked Desialylation of Cell Surface Glycans: Implications for Ischemia-Targeted Gene Therapy

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Arabindra B Katwal ◽  
Eric T Mulkey ◽  
R John Lye ◽  
Brent A French ◽  
Brian H Annex
Keyword(s):  
Skeletal Muscle ◽  
Gene Therapy ◽  
Peripheral Arterial Disease ◽  
Cell Surface ◽  
Limb Ischemia ◽  
Arterial Disease ◽  
Fluorescence Signal ◽  
Differential Distribution ◽  
Peripheral Arterial ◽  
Cell Surface Glycans

Introduction: Ischemic skeletal muscle is preferentially targeted after IV injection of AAV9 in mouse models of hind-limb ischemia (HLI). Ischemia is known to induce desialylation of cell surface glycans, unmasking the cellular receptor for AAV9 and resulting in significantly higher levels of selective gene expression in ischemic vs non-ischemic muscle. To assess clinical relevance, we compared the differential distribution of sialylated and desialylated cell surface glycans in skeletal muscle biopsies from peripheral arterial disease (PAD) patients and normal controls. Methods: Gastrocnemius (GA) muscle samples from PAD patients and normals (n=29 each) were biopsied under IRB-approved protocols. Muscle sections were stained using the lectins MAL-I and ECL. MAL-I binds to α2,3-sialylated glycans whereas ECL binds to desialylated galactose residues on cell surface glycans. Lectins were labeled with Streptavidin-Alexa-488 (red) for MAL-I and SA-555 (green) for ECL, and visualized using fluorescence microscopy. Quantification of mean fluorescence signal intensity of lectin staining was performed on each section. Results: Visually, MAL-I staining was markedly higher and ECL staining lower in non-ischemic GA from normals when compared to ischemic GA from PAD patients (Figure). The ratio of MAL-I to ECL integrated fluorescence intensity in the ischemic muscles from PAD patients (1.13 ± 0.13) was significantly lower than the same ratio in non-ischemic muscles from normals (1.58 ± 0.14, p<0.02). Conclusions: Desialylation of cell surface glycans is significantly higher in ischemic skeletal muscle from PAD patients, consistent with previous work in a mouse model of HLI. This desialylation can potentially be exploited to treat human PAD with gene therapy, since these desialylated cell surface glycans serve as cell surface receptors for AAV9 and provide for efficient and selective gene transfer to ischemic skeletal muscle following systemic or local delivery.

Pre-Clinical Studies of Therapeutic Angiogenesis Induced by Human Hepatocyte Growth Factor (HGF) Gene in Rat and Rabbit Hind Limb Ischemia Models

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 706-706
Author(s):  
Yoshiaki Taniyama
Keyword(s):  
Gene Therapy ◽  
Peripheral Arterial Disease ◽  
Intramuscular Injection ◽  
Hind Limb ◽  
Limb Ischemia ◽  
Arterial Disease ◽  
Therapeutic Angiogenesis ◽  
Hindlimb Ischemia ◽  
Angiogenic Growth Factors ◽  
Peripheral Arterial

P72 A novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy. Initially, we examined the transfection of “naked” human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imager. Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (C100% vs H 245%, P<0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hind limb models, as potential therapy for peripheral arterial disease.

Gene therapy for therapeutic angiogenesis in peripheral arterial disease - a systematic review and meta-analysis of randomized, controlled trials

VASA ◽  
2013 ◽  
Vol 42 (5) ◽  
pp. 331-339 ◽  
Author(s):  
Alexandra Hammer ◽  
Sabine Steiner
Keyword(s):  
Gene Therapy ◽  
Peripheral Arterial Disease ◽  
Meta Analysis ◽  
Limb Ischemia ◽  
Arterial Disease ◽  
Therapeutic Angiogenesis ◽  
Controlled Trials ◽  
Randomized Controlled ◽  
All Cause Mortality ◽  
Peripheral Arterial

Background: Beyond pharmacological, endovascular and surgical treatment strategies for peripheral arterial disease (PAD), therapeutic angiogenesis has been advocated to relieve symptoms and support limb salvage, in particular in patients with critical limb ischemia. We aimed to systematically review randomized controlled trials (RCTs) of gene therapy in PAD. Patients and methods: A systematic search of electronic databases was performed to identify RCTs studying local administration of pro-angiogenic growth factors (VEGF, FGF, HGF, Del-1, HIF-1alpha) using plasmid or viral gene transfer by intra-arterial or intra-muscular injections. Outcomes of interest comprised all-cause mortality, amputations, ulcer healing, walking distance and ankle-brachial index. If feasible, standard meta-analysis should be performed with subgroup analysis for claudicants and patients with critical limb ischemia (CLI). Results: The systematic search yielded 12 RCTs for analysis from 1163 citations. In total, 1494 patients (29 % females) were included with the majority suffering from CLI (64 %). Various endpoints were improved by single studies, but none by a majority of studies. Meta-analysis showed neither a significant benefit nor harm for gene therapy when synthesizing data for all-cause mortality (OR 0.88, 95 % CI 0.62 - 1.26) amputations (OR 0.64, 95 % CI 0.31 - 1.31) or ulcer healing (OR 1.79, 95 % CI 0.8 - 4.01). No differences were seen between patients with intermittent claudication or CLI. Conclusions: Despite promising results in single studies, no clear benefit could be identified for gene therapy in PAD patients, irrespective of disease severity.

Abstract 20: Resolution Phenotype of Monocytes and Macrophages is Altered in Peripheral Arterial Disease

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Melinda S Schaller ◽  
Laura Menke ◽  
Mian Chen ◽  
Warren J Gasper ◽  
S. Marlene Grenon ◽  
...  
Keyword(s):  
Peripheral Arterial Disease ◽  
Cell Surface ◽  
Phagocytic Activity ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Arterial Disease ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Peripheral Blood Mononuclear ◽  
Peripheral Arterial

Introduction: Peripheral arterial disease (PAD) is a chronic disease characterized by systemic inflammation. Monocytes (Mo) and macrophages play a central role in vascular inflammation and its resolution. We hypothesize that impaired resolution in PAD results in poor clinical outcomes. Methods: Resolution phenotype was assessed by phagocytic activity of leukocytes, Mo cell surface markers, and cytokine profiling of Mo-derived macrophages (MDM). Phagocytosis and cell-surface markers were determined by flow cytometry. MDMs were generated from peripheral blood mononuclear cells via density gradient centrifugation. Cytokines were measured by ELISA following MDM differentiation and subsequent stimulation with LPS. Results: Circulating Mo and neutrophils (PMN) isolated from PAD patients (n=9) demonstrated significantly lower phagocytic activity (Mo: >30%, p<.001; PMN: >25%, p<.01, Fig. 1) as compared to healthy subjects (HS) (n=14). Cell-surface marker analysis demonstrated a higher proportion of the pro-inflammatory intermediate Mo subset (CD14 ++ 16 + , 1.8-fold, p=.04) in PAD compared to HS. MDM from PAD subjects retain their intrinsic inflammatory program by producing more IL-6 (PAD 3138±2676 ng/mL, HS 731±854 ng/mL p=.03) and IL-1β (PAD 244±236 ng/mL, HS 24.1±23.8 ng/mL p=.04) than those from HS. Upon stimulation with LPS, MDM from PAD subjects secrete more IL-6 (PAD 23353±22483 ng/mL, HS 5097±5836 ng/mL p=.05) than those from HS. Conclusions: Circulating Mo and PMN in patients with PAD have substantially lower phagocytic activity as well as a greater proportion of the pro-inflammatory intermediate Mo subset compared to HS. MDM preserve their elevated inflammatory state throughout culture and retain a heightened response upon latter stimulatory cues. Collectively these data demonstrate a heightened inflammatory and impaired resolution phenotype in PAD that has potential implications for disease progression and response to interventions.

Abstract P250: Incidence of Hospitalized Peripheral Arterial Disease and Critical Limb Ischemia: The Atherosclerosis Risk in Communities (ARIC) Study

Circulation ◽  
2012 ◽  
Vol 125 (suppl_10) ◽  
Author(s):  
Corey A Kalbaugh ◽  
Anna Kucharska-Newton ◽  
Laura Loehr ◽  
Elizabeth Selvin ◽  
Aaron R Folsom ◽  
...  
Keyword(s):  
Risk Factors ◽  
African Americans ◽  
Peripheral Arterial Disease ◽  
Lower Extremity ◽  
Limb Ischemia ◽  
Arterial Disease ◽  
Atherosclerosis Risk In Communities ◽  
Atherosclerosis Risk ◽  
Baseline Characteristics ◽  
Peripheral Arterial

Introduction: Lower extremity peripheral arterial disease (PAD) affects between 12% and 20% of Americans over the age of 65. PAD compromises quality of life, contributes a high burden of disability and its related health care costs exceed $4 billion/year, yet this preventable CVD outcome remains understudied. Aims: Assess the incidence of hospitalized PAD, and of the most severe form of PAD, critical limb ischemia (CLI), in middle-aged men and women, and evaluate their risk factors in a bi-ethnic, population-based cohort. We hypothesized that incidence of hospitalized PAD and CLI are higher in African Americans, and that modifiable atherosclerosis risk factors in middle age predict these sequelae of PAD. Methods: We analyzed data from 13,865 participants from the Atherosclerosis Risk in Communities Study aged 45–64 without PAD at baseline (1987–89). Incident PAD and CLI events were identified using ICD-9 codes from active surveillance of all hospitalizations among cohort participants from 1987 through 2008. All estimates are incidence rates per 10,000 person-years; nominal statistical significance was achieved for all baseline characteristic comparisons reported. Results: There were 707 incident hospitalized PAD during a median of 18 years of follow-up (249,570 person-years). The overall age-adjusted incidence of PAD and limb-threatening CLI were 26.0 and 9.6 per 10,000 person-years, respectively. Incidence of hospitalized PAD was higher in African Americans than whites (34.7 vs. 23.2) and in men compared to women (32.4 vs. 26.7). Baseline characteristics associated with age-adjusted incident PAD (per 10,000 person-years) compared to their referent groups were diabetes (91.2 vs. 19.0), history of smoking (33.6 vs. 16.2), hypertension (42.6 vs. 18.6), coronary heart disease (81.4 vs. 24.1), and obesity (41.5 vs. 20.2). Incidence of CLI also was higher among African Americans (21.0 vs. 5.9) and in men (10.5 vs. 8.9 per 10,000 person-years). Baseline characteristics associated with incident CLI were similar to those for PAD. Conclusions: The absolute risk of hospitalized lower extremity PAD in this community-based cohort is of a magnitude similar to that of heart failure and of stroke. As modifiable factors are strongly predictive of the long-term risk of hospitalized PAD and CLI, particularly among African Americans, our results highlight the need for effective risk factor prevention and control.

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