Critical Limb Ischemia In Diabetic Patients Research Articles

Abstract 249: Plasma Exosomes Impair Angiogenesis in Ischemic Hind Limb of Diabetic Mice- Role of Histone Methylation

Background: Critical limb ischemia (CLI) is one of most prevenient cardiovascular disease in diabetic patients. Recent evidence suggests that altered cargo and function of plasma exosomes (plasma-Exo) may play an important role in diabetes-induced cardiovascular complications. Here, we tested the hypotheses that inhibition of exosome biosynthesis/release improves ischemic hind limb (IHL) repair in db/db mice. Methods: Plasma-Exo from db/+ and db/db mice were isolated by density-gradient ultracentrifugation. Unilateral IHL in mice was conducted by ligation of left femoral artery. Blood perfusion in IHL was measured by Laser Doppler Imager. Results: Diabetic plasma-Exo impaired tube formation/migration of human microvascular endothelial cells (HMVECs) and blood perfusion in IHL of C57BL/6J mice. Exosome inhibitor GW4869 improved blood flow, capillary density, cell survival, and rescued necrosis of toe/toenail and fibrosis in IHL muscle of db/db mice. Mechanistically, diabetic plasma-Exo decreased secretion of pro-angiogenic factor Ang I&II, artemin, FGF2 and IGFBP1&2, and increased repressive transcriptional mark H3K27me3 and its methylase enhancer of zest homolog-2 (EZH2) in HMVECs. EZH2 inhibitor GSK343 rescued diabetic plasma-Exo-impaired tube formation and secretion of FGF2/artemin from HMVECs. Moreover, GW4869 reduced EZH2 and H3K27me3 protein expression in lung microvascular ECs of IHL db/db mice. Finally, diabetic plasma-Exo increased H3K27me3 level at promoter of artemin and FGF2. Conclusions: Diabetic plasma-Exo impair angiogenesis and IHL injury repair. Diabetic plasma-Exo impair reparative property of ECs via, at least in part, enhancement of EZH2/H3K27me3/artemin and FGF2 cascade. Inhibition of plasma-Exo biosynthesis/secretion improve IHL repair in db/db mice. Plasma-Exo may be a novel target for prevention/treatment of CLI in diabetic patients.

Abstract 23: Exosome Inhibition Improved Blood Perfusion in Ischemic Hindlimb of db/db Diabetic Mice

Background: Critical limb ischemia (CLI), a life-threatening condition characterized by pain at rest and tissue loss with ulcer and gangrene, imposes a major public healthy burden, resulting in high mortality and disability. The occurrence of CLI in patients with diabetes mellitus is very frequent. However, the effective therapy for CLI in diabetic patients is absent. Recent studies demonstrated that exosome from diabetic animals/cells has detrimental effects on the post-injury cardiovascular repair. Here, we tested the hypotheses that exosome inhibition in vivo improves blood flow recovery and protects skeletal muscle in ischemic hindlimbs of diabetic db/db mice following surgical ischemia. Methods and Results: Exosomes were isolated from bone-marrow derived progenitor cells or plasma in non-diabetic db/+ and diabetic db/db mice by ultracentrifugation. Diabetic exosome (5 ug/ml) inhibited tube formation of human cardiac microvascular endothelial cells. Unilateral hindlimb ischemia surgery was conducted by ligation of left femoral artery in 12-week old, male db/+ and db/db mice. Exosome inhibitor GW4869 (2 μg/g body weight) was given by intraperitoneal injection every other day for 4 weeks starting from one week before the HLI surgery. HLI mice injected with vehicle served as controls. Mice were divided into four groups: 1) db/+ + vehicle; 2) db/db+ vehicle; 3) db/+ GW4869; 4) db/db + GW4869. GW4869 decreased necrosis and loss of toe/toenail, improved blood flow, enhanced capillary/arterial density, skeletal muscle architecture and cell survival in ischemic hindlimb of diabetic db/db mice 21 days post-ligation. Conclusions: Although preliminary, our experiments suggest that therapeutic targeting of dysfunctional exosome secretion could represent a new avenue for the prevention and treatment of ischemic injury in diabetic patients.

Long-term prognosis of diabetic patients with critical limb ischemia after endovascular therapy

Introduction. Critical limb ischemia (CLI) occurs approximately 20 times more frequently among diabetic patients. Frequency of amputations is higher too. Main treatment goal in these situations is in reconstruction of arterial blood flow with bypass surgery or endovascular interventions. Nevertheless, long-term prognosis of survival, limb preservation and life quality among Russian patients that underwent this therapy remains unclear, as well as influencing risk factors.The aim of the study was to evaluate the long-term prognosis in diabetic patients with critical limb ischemia (CLI) after peripheral angioplasty with active and nonactive follow-up period.Methods. 81 diabetic patients with CLI underwent PTA in 88 limbs. Patients were divided into 2 groups: group A (n = 51) – with active follow up (FU) period (visits every 3-6 months during 5 years) and group B (n = 30) - without active FU period (the second visit in our center was performed in 5 years after PTA). Diagnosis and treatment of CLI were based on recommendation of TASC II. The primary outcome was cumulative survival, secondary outcome were cases of repeat PTA and major amputations (MA) after 5 years after surgery.Results. Only 44 (86%) patients from group A finished FU period. There were 37 (46%) men, with mean age 64,1[54-68] years, mean HbA1c 7,9±1,4%, mean duration of diabetes 16,5[0,8-43] years, diabetes type 1/2 - 8/73 (90% of type 2). 82% had arterial hypertension, 5% - strokes, 18,5% - myocardial infarction. Chronical kidney disease of stage 1-2 was detected in 55,5%, stage 3-5 – in 30,8%. Anemia was diagnosed in 67,5%, arrhythmias – in 7,4%. 49,3% of patients suffered from diabetic retinopathy. Patients from both groups were comparable in comorbidities, severity of lower limb artery obstruction’s and degree of tissue damage (p<0,05): peripheral arterial disease (PAD) 4-6 classes according Graziani classification in both groups was in 75(93%) cases; Rutherford classification in both groups: 4 category-12(15%), 5category in 43(53%), and 6 category in 29(31%) patients. Repeat PTA was performed in group A in 15 (35%), in group B in 5(16%) cases. There were major amputations in groups: A-4(9%) vs group B – 4(12%) (log-rank, p<0,05). Cumulative survival in groups: A-80%, in group B-67%. (log-rank, p<0,05).Conclusion. CLI in diabetic patients is accompanied by different complications and is characterized by severe morphological lesions of the lower limbs arteries and soft tissue lesions. Active FU period have advantages in diabetic patients with CLI after PTA: timely done reinterventions with decrease the risk of major amputations and cumulative survival.

Long-Term Clinical Outcomes of Infrapopliteal Drug-Eluting Stent Placement for Critical Limb Ischemia in Diabetic Patients

PurposeTo evaluate long-term clinical outcomes of infrapopliteal drug-eluting stent (DES) placement in insulin-dependent and non–insulin-dependent diabetic patients with critical limb ischemia (CLI). Materials and MethodsA retrospective analysis was performed of all diabetic patients treated with infrapopliteal DES between January 2002 and September 2012. The study’s primary outcome measures were patient survival and major amputation–free survival (AFS). Secondary outcome measures included technical success (defined as the creation of a straight line of blood flow to the foot arch with < 30%), identification of independent predictors of primary outcomes, infrapopliteal target limb repeat intervention–free survival, and procedure-related complications. ResultsIn total, 214 patients with CLI (168 men [78.5%]; mean age, 70 y ± 9) in 311 limbs, 562 arteries, and 679 lesions were treated. According to Kaplan–Meier analysis, survival rates were 90.8%, 55.5%, and 36.2%, and AFS rates were 94.9%, 90.4%, and 90.4%, respectively, at 1, 5, and 10 years. Target limb repeat intervention–free survival rates were 79.7%, 55.2%, and 49.7%, respectively, at 1, 5, and 10 years. The overall technical success rate was 97.7%. Cox multivariate analysis demonstrated that procedural failure was the only independent predictor of decreased AFS (hazard ratio [HR], 61.3; 95% confidence interval [CI], 13.8–271.9), and statin use was associated with increased survival (HR, 0.55; 95% CI, 0.31–0.98). Coronary disease (HR, 1.9; 95% CI, 1.01–3.54), dialysis (HR, 2.2; 95% CI, 1.21–4.06), and duration of diabetes (HR, 1.5; 95% CI, 1.02–2.34) were identified as independent predictors of decreased survival. Major complications occurred in four of 479 procedures (0.8%). ConclusionsInfrapopliteal DES placement for the management of CLI in diabetic patients resulted in a 55.5% 5-year survival rate, with a 90.4% AFS at 5 and 10 years and a 50.3% repeat intervention rate at 10 years. Technical failure was associated with reduced AFS, and statin intake was associated with increased survival.

Abstract 18084: Diabetes Promotes Tissue Nitric Oxide (NO) Insufficiency in Critical Limb Ischemia Patients

Background: Diabetic patients are 5 times more likely than non-diabetics to develop critical limb ischemia (CLI). The pathogenesis of CLI in diabetic patients remains poorly understood and at present there is a paucity of effective therapies to treat this devastating disease. Nitrite (NO2) and nitrosothiols (RSNO) have recently emerged as important storage forms of nitric oxide (NO) that modulate vascular reactivity and signaling. The purpose of this study was to investigate tissue NO bioavailability in muscle biopsies obtained from non-diabetic and diabetic (Db) patients following leg amputation. Methods: Gastrocnemius and tibialis anterior tissue was attained post-amputation from human non-diabetic patients with CLI (n=12) and diabetic patients with CLI (n=8). Nitrite concentrations were determined by ion chromatography methods. RSNO and NO-Heme levels were measured by chemiluminescence detection. Results: The CLI + Db group displayed reduced skeletal muscle (89.9 vs. 55.5 nM, p &lt; 0.05) nitrite levels compared to the CLI group without Db. Nitrosothiol (21.2 vs. 10.52 nM, p &lt; 0.05) concentrations and NO-Heme (27.61 vs. 13.0 nM, p &lt; 0.05) levels were also significantly attenuated in the CLI + Db group. Conclusion: These data clearly indicate significant reductions in NO bioavailability (nitrite, RSNO, and NO-heme) in diabetics with CLI. Future studies will evaluate the cellular mechanisms responsible for reduced NO levels in diabetic patients with CLI. Nitric oxide based therapeutics including sodium nitrite may be suitable to treat diabetic patients with CLI to attenuate the progression of this disease.

Role of Ultrasound Arterial Mapping in Planning Therapeutic Options for Critical Ischemia of Lower Limbs in Diabetic Patients

To assess the role of ultrasound arterial mapping in planning therapeutic options for critical limb ischemia (CLI) in diabetic patients. This observational and comparative study included 244 patients with CLI. All participants (64% were diabetic) underwent ultrasound arterial mapping before planning surgical treatment. We established two groups: diabetic (n = 156) and nondiabetic (n = 88). Arterial mapping was divided into segments. We studied 2,021 individual segments and used arteriography when ultrasound arterial mapping was not conclusive. We compared the degree of pathology between the groups, agreement between the treatment decision made after ultrasound mapping and the final surgical decision in both groups, and agreement between ultrasound mapping and arteriography in patients who underwent both procedures. Diabetic patients had a significantly higher degree of pathology in all segments, except the common iliac artery. Decisions made after ultrasound mapping matched the final surgical decision 90% and 94% of the time in diabetic patients and nondiabetic patients, respectively. Decisions made on the basis of ultrasound arterial mapping matched decisions made on the basis of arteriography in 86.3%. Ultrasound arterial mapping allowed for good therapeutic planning for CLI in diabetic patients, even though these patients had more severe arterial pathology.