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OBJECTIVE: To evaluate the impact of N-acetyl-cysteine (NAC) on amputation stump perfusion and healing in patients with critical limb-threatening ischemia (CLTI) BACKGROUND: Patients with CLTI are at increased risk of poor amputation site healing leading to increased procedure-associated morbidity. METHODS: In a pilot, double-blind, placebo-controlled, randomized controlled trial, patients with CLTI undergoing major elective lower extremity amputation were randomized 1:1 to intravenous NAC (1200mg twice-daily) or placebo for up to 5 days postoperatively. Primary outcomes were change in stump perfusion at postoperative days 3 (POD3) and POD5, and healing at POD30. Stumps were serially evaluated for wound healing, and tissue perfusion was evaluated using non-invasive Laser-Assisted Fluorescent Angiography (LAFA). RESULTS: Thirty-three patients were randomized to NAC (n=16) or placebo (n=17). 31 patients were eligible for intent-to-treat analysis (NAC14; placebo17). 20 patients (NAC7; placebo13) had amputation stump perfusion defects at POD0 and were considered high-risk for poor healing. Intent-to-treat analysis revealed no significant differences between treatment groups. Subgroup analysis of high-risk patients revealed differences in stump perfusion defect size (NAC −0.53-fold, placebo +0.71-fold; 95% CI −2.11 to −0.35; p

Acute deep vein thrombosis (DVT) is a potentially life-threatening condition in which blood clots form in the venous system. Recently, a number of endovascular thrombectomy devices have been commercialized for the treatment of acute DVT. With variable efficacy and safety profiles, the current state-of-the-art has identified potential areas for further device innovation and opportunities for improvement. However, a major limitation in the testing, verification, and validation of new thrombectomy is a clinically representative large animal model of acute DVT. Unfortunately, existing approaches to creating DVT in porcine models are time consuming, technically challenging, and provide inconsistent results. We therefore developed a rapid and more reproducible protocol for induction of large-volume iliocaval DVTs in a living porcine host. This approach involves sequestering the iliocaval with standard balloon occlusion catheters, and a controlled and limited infusion of a 25% ethanol solution within the sequestered segment. We observed that this method was safe, technically straightforward, and reproducible in creating large-volume acute DVTs. We believe this model can be utilized in a standardized approach for the future testing of future endovascular venous thrombectomy devices.

Objectives: Chemokines and their receptors play critical roles in the progression of atherosclerosis. The chemokine receptor, C-C chemokine receptor type 2 (CCR2), mediates an essential role in the differentiation of pro-inflammatory monocytes into macrophages. Recent studies suggest that proinflamatory macrophages in plaques predict plaque progression. While there is no clinical consensus on which patient with asymptomatic carotid artery stenosis would benefit from carotid endarterectomy (CEA), we hypothesized that CCR2 plaque content may predict plaque progression. Methods: Carotid plaque of ten patients undergoing elective CEA were harvested from the operating room, paraffin-embedded, sectioned into 5μm segments, immuno-stained for CCR2 and CD68, and imaged. Three 200х500μm regions-of-interest (ROIs) were randomly selected within 200μm of the lumen (superficial intima), outside 200μm of the lumen (deep intima), and the arterial media. Two independent observers evaluated the number of CCR2 and/or CD68 positive cells. Counts in each ROI were averaged and analyzed with Student’s t-test. Results: Patients with symptomatic carotid artery stenosis (6/10) demonstrated higher CCR2 and CCR2-CD68 double positive cells within the superficial intima of carotid artery plaques (A, p Conclusions: Our study demonstrates the close association between CCR2 cellular content and the incidence of symptomatic carotid artery disease. Additionally, CCR2 plaque content appears to correlate with risk-factors such as smoking. These studies have important implications regarding the impact of immune modulation on carotid plaque vulnerability in patients with asymptomatic carotid artery stenosis.

Objectives: Choline enthanolamine phosphotransferase 1 ( CEPT1 ), is essential for endothelial de novo lipogenesis (DNL) and is elevated in diseased peripheral arterial segments. Women are more sensitive to diet-induced DNL, and those with diabetes have higher incidence of severe peripheral arterial disease (PAD). We therefore hypothesized that women may have higher CEPT1 content in diseased peripheral arteries and that murine conditional endothelial overexpression of CEPT1 may lead to sex-specific differences in ischemic recovery. Methods: CEPT1 expression was evaluated in diseased peripheral plaque of 7 women and 9 men. A murine endothelial CEPT1 overexpression model was engineered with Cre-induced expression of a CEPT1 transgene inserted in a C57BL/6J background. Endothelial CEPT1 overexpression was evaluated using RT-PCR. Male (n=5) and female (n=5) received Streptozotocin and unilateral femoral artery ligation. Limb perfusion, appearance, and use were then evaluated. Gastrocnemius was stained with H&E, and endothelial Isolectin. Muscle fiber size and microvascular density were analyzed using Student’s t-test. Results: Compared to males, female patients have higher CEPT1 in peripheral segments (n=7, pCEPT1 overexpression had a 7-fold increase in expression compared to control (Fig B). Compared to female mice, males demonstrated improved perfusion (p Conclusion: This study reveals a gender difference in peripheral arterial CEPT1 expression and ischemic recovery following overexpression. Differences do not appear to be directly associated with angiogenesis and may be due to underlying DNL metabolic consequences. These findings may have important implications for the prevalence of severe PAD in diabetic women.

De novo phospholipogenesis, mediated by choline-ethanolamine phosphotransferase 1 (CEPT1), is essential for phospholipid activation of transcription factors such as peroxisome proliferator–activated receptor α (PPARα) in the liver. Fenofibrate, a PPARα agonist and lipid-lowering agent, decreases amputation incidence in patients with diabetes. Because we previously observed that CEPT1 is elevated in carotid plaque of patients with diabetes, we evaluated the role of CEPT1 in peripheral arteries and PPARα phosphorylation (Ser12). CEPT1 was found to be elevated in diseased lower-extremity arterial intima of individuals with peripheral arterial disease and diabetes. To evaluate the role of Cept1 in the endothelium, we engineered a conditional endothelial cell (EC)–specific deletion of Cept1 via induced VE-cadherin-CreERT2–mediated recombination (Cept1Lp/LpCre+). Cept1Lp/LpCre+ ECs demonstrated decreased proliferation, migration, and tubule formation, and Cept1Lp/LpCre+ mice had reduced perfusion and angiogenesis in ischemic hind limbs. Peripheral ischemic recovery and PPARα signaling were further compromised by streptozotocin-induced diabetes and ameliorated by feeding fenofibrate. Cept1 endoribonuclease-prepared siRNA decreased PPARα phosphorylation in ECs, which was rescued with fenofibrate but not PC16:0/18:1. Unlike Cept1Lp/LpCre+ mice, Cept1Lp/LpCre+Ppara−/− mice did not demonstrate hind-paw perfusion recovery after feeding fenofibrate. Therefore, we demonstrate that CEPT1 is essential for EC function and tissue recovery after ischemia and that fenofibrate rescues CEPT1-mediated activation of PPARα.

ObjectivePeripheral atherosclerosis that accumulates in the extracranial carotid and lower extremity arteries can lead to significant morbidity and mortality. However, atherosclerotic disease progression is often not homogenous and is accelerated by diabetes. We previously observed increased phospholipid content in minimally (Min)-diseased arterial segments compared to maximally (Max)-diseased segments. Since Peroxisome Proliferator-Activated Receptor alpha (PPARα) is a key regulator of lipid metabolism, we hypothesized that it may have differential expression and signaling in Min vs. Max-diseased peripheral arterial segments.MethodsEighteen patients who underwent carotid endarterectomy (CEA), and 34 patients who underwent major lower extremity amputation were prospectively enrolled into a vascular tissue biobank. Min and Max-diseased segments were obtained in real-time from CEA plaque and amputated lower extremity arterial segments. mRNA and protein were isolated from specimens and the relative expression of ppara, and its downstream genes Acyl-CoA Oxidase 1 (acox1) and Carnitine Palmitoyltransferase 1A (cpt1a) were also evaluated. We evaluated gene expression and protein content relative to atherosclerotic disease severity and clinical diabetes status. Gene expression was also evaluated relative to Hemoglobin A1c and serum lipid profiles.ResultsIn CEA segments of patients with diabetes, we observed significantly higher ppara and acox1 gene expression (p < 0.01 and p < 0.001 respectively), and higher PPARα protein content (p < 0.05). Hemoglobin A1c significantly correlated with expression of ppara (R2 = 0.66, p < 0.001), acox1 (R2 = 0.31, p < 0.05), and cpt1a (R2 = 0.4, p < 0.05). There was no significant difference in gene expression between Min vs. Max-diseased CEA plaque segments. Conversely, in lower extremity arterial segments of patients with diabetes, we observed significantly lower ppara, acox1, and cpt1a expression (p < 0.05, p < 0.001, and p < 0.0001 respectively). Interestingly, CPT1A content was lower in arterial segments of patients with diabetes (p < 0.05). Hemoglobin A1c and HDL-cholesterol had negative correlations with ppara (R2 = 0.44, p < 0.05; R2 = 0.42, p < 0.05; respectively).ConclusionThis study demonstrates the significant differential expression of ppara and its immediate downstream genes in human carotid and lower extremity arteries relative to disease severity and diabetes. These findings highlight that mechanisms that influence atheroprogression in the carotid and lower extremities peripheral arteries are not homogenous and can be impacted by patient diabetes status and serum cholesterol profiles. Further elucidating these differential molecular mechanisms can help improve targeted therapy of atherosclerosis in different peripheral arterial beds.

There are currently no serum-based evaluations that can corroborate the severity of peripheral artery disease (PAD). In this cross-sectional study, we assessed the prevalence of elevated serum fatty acid synthase (cFAS) in patients with chronic limb-threatening ischemia (CLTI) and evaluated the accuracy of its use in detecting this condition. Preoperative fasting serum samples from 87 patients undergoing vascular intervention were collected between October 2014 and September 2016. Median age was 62 years, with 56 (64%) men, and 32 (37%) with CLTI. We found that elevated cFAS content (OR 1.17; 95% CI 1.04–1.31), type 2 diabetes (T2D; OR 5.22; 95% CI 1.77–15.4), and smoking (OR 3.53; 95% CI 1.19–10.5) were independently associated with CLTI and could detect the presence of CLTI with 83% accuracy (95% CI 0.74–0.92). Furthermore, serum FAS content was positively correlated with FAS content in femoral artery plaque in patients with severe PAD ($${R}^{2}$$ R 2 = 0.22; P = 0.023). Finally, significantly higher co-localization of FAS and ApoB were observed within lower extremity arterial media (P

Peripheral atherosclerosis manifests in both the extracranial carotid and lower extremity arteries and can lead to significant morbidity and mortality. However, atherosclerotic disease progression is often not homogenous and is accelerated by diabetes. We previously observed altered phospholipodomic profiles between minimally (MIN) and maximally (MAX) diseased peripheral arterial segments. Since Peroxisome Proliferator-Activated Receptor alpha ( pparα ) is a key regulator of lipid metabolism, we hypothesized that it may have variable content and signaling in MIN and MAX diseased arterial segments. To test our hypothesis, 12 patients who underwent carotid endarterectomy (CEA), and 19 patients who underwent major lower extremity amputation were recruited. MIN and MAX disease segments were obtained in real time from the operating room from CEA plaque and arterial segments from amputated lower extremities. mRNA was isolated from all specimens and relative content of pparα , Acyl-CoA Oxidase 1 ( acox1 ) , and Carnitine Palmitoyltransferase 1A ( cpt1a ) were evaluated. We observed significantly higher pparα expression in CEA segments in patients with diabetes (p < 0.01), as well as higher acox1 (p < 0.001) and cpt1a (p < 0.05) expression (A-C). Hemoglobin A1C had a significant correlation with ppara gene expression. There was no significant difference in gene expression between MAX and MIN diseased CEA plaque segments. Interestingly, we observed that in lower extremity arterial segments there was no difference in ppara , acox1 , and cpt1a in patients with and without diabetes, but downstream genes were significantly increased in MAX diseased arterial segments (D-F). This study demonstrates the variable expression pattern of pparα and its downstream genes in human peripheral arteries. Our findings suggest variable gene expression in different peripheral arterial beds, which may have an impact on mechanisms of disease progression and pharmacologic targeting.

De novo phosopholipogenesis, mediated by choline-ethanolamine phosphotransferase 1 (CEPT1), is essential for phospholipid activation of transcription factors such as peroxisome proliferator-activated receptor α (PPARα) in the liver. Fenofibrate, a PPARα agonist and lipid-lowering agent, decreases amputation incidence in patients with diabetes. Since we previously observed that CEPT1 is elevated in carotid plaque of patients with diabetes, we evaluated the role of CEPT1 in peripheral arteries and PPARα-phosphorylation (Ser12). CEPT1 was found to be elevated in diseased lower extremity arterial intima of individuals with peripheral arterial disease and diabetes. To evaluate the role of Cept1 in the endothelium, we engineered a conditional endothelial cell (EC)-specific deletion of Cept1 via induced VE-cadherin-CreERT2 mediated recombination (Cept1Lp/LpCre+). Cept1Lp/LpCre+ ECs demonstrated decreased proliferation, migration, and tubule formation, and Cept1Lp/LpCre+ mice had reduced perfusion and angiogenesis in ischemic hind-limbs. Peripheral ischemic recovery and PPARα signaling was further compromised by Streptozotocin-induced diabetes, and ameliorated by feeding fenofibrate. Cept1 esiRNA decreased PPARα-phosphorylation in ECs, which was rescued with fenofibrate but not PC16:0/18:1. Unlike Cept1Lp/LpCre+, Cept1Lp/LpCre+Ppara-/- mice did not demonstrate hind-paw perfusion recovery after feeding fenofibrate. Therefore we demonstrate that CEPT1 is essential for EC function and tissue recovery following ischemia, and that fenofibrate rescues CEPT1-mediated activation of PPARα.