Nicolas Diehm

Novel cell-free strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation

Background Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. Methodology/Principal Findings EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34+/CD45− cells/1×105 mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34+ cells/HPF, P<0.001) 3 days after the last injection. Conclusions/Significance Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.

Fixed low-dose ultrasound-assisted catheter-directed thrombolysis for intermediate- and high-risk pulmonary embolism

AIMS No standardized local thrombolysis regimen exists for the treatment of pulmonary embolism (PE). We retrospectively investigated efficacy and safety of fixed low-dose ultrasound-assisted catheter-directed thrombolysis (USAT) for intermediate- and high-risk PE. METHODS AND RESULTS Fifty-two patients (65 ± 14 years) of whom 14 had high-risk PE (troponin positive in all) and 38 intermediate-risk PE (troponin positive in 91%) were treated with intravenous unfractionated heparin and USAT using 10 mg of recombinant tissue plasminogen activator per device over the course of 15 h. Bilateral USAT was performed in 83% of patients. During 3-month follow-up, two [3.8%; 95% confidence interval (CI) 0.5-13%] patients died (one from cardiogenic shock and one from recurrent PE). Major non-fatal bleeding occurred in two (3.8%; 95% CI, 0.5-13%) patients: one intrathoracic bleeding after cardiopulmonary resuscitation requiring transfusion, one intrapulmonary bleeding requiring lobectomy. Mean pulmonary artery pressure decreased from 37 ± 9 mmHg at baseline to 25 ± 8 mmHg at 15 h (P < 0.001) and cardiac index increased from 2.0 ± 0.7 to 2.7 ± 0.9 L/min/m(2) (P < 0.001). Echocardiographic right-to-left ventricular end-diastolic dimension ratio decreased from 1.42 ± 0.21 at baseline to 1.06 ± 0.23 at 24 h (n = 21; P < 0.001). The greatest haemodynamic benefit from USAT was found in patients with high-risk PE and in those with symptom duration < 14 days. CONCLUSION A standardized catheter intervention approach using fixed low-dose USAT for the treatment of intermediate- and high-risk PE was associated with rapid improvement in haemodynamic parameters and low rates of bleeding complications and mortality.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Chapter IV: Treatment of Critical Limb Ischaemia

Recommendations stated in the TASC II guidelines for the treatment of peripheral arterial disease (PAD) regard a heterogeneous group of patients ranging from claudicants to critical limb ischaemia (CLI) patients. However, specific considerations apply to CLI patients. An important problem regarding the majority of currently available literature that reports on revascularisation strategies for PAD is that it does not focus on CLI patients specifically and studies them as a minor part of the complete cohort. Besides the lack of data on CLI patients, studies use a variety of endpoints, and even similar endpoints are often differentially defined. These considerations result in the fact that most recommendations in this guideline are not of the highest recommendation grade. In the present chapter the treatment of CLI is not based on the TASC II classification of atherosclerotic lesions, since definitions of atherosclerotic lesions are changing along the fast development of endovascular techniques, and inter-individual differences in interpretation of the TASC classification are problematic. Therefore we propose a classification merely based on vascular area of the atherosclerotic disease and the lesion length, which is less complex and eases the interpretation. Lesions and their treatment are discussed from the aorta downwards to the infrapopliteal region. For a subset of lesions, surgical revascularisation is still the gold standard, such as in extensive aorto-iliac lesions, lesions of the common femoral artery and long lesions of the superficial femoral artery (>15 cm), especially when an applicable venous conduit is present, because of higher patency and limb salvage rates, even though the risk of complications is sometimes higher than for endovascular strategies. It is however more and more accepted that an endovascular first strategy is adapted in most iliac, superficial femoral, and in some infrapopliteal lesions. The newer endovascular techniques, i.e. drug-eluting stents and balloons, show promising results especially in infrapopliteal lesions. However, most of these results should still be confirmed in large RCTs focusing on CLI patients. At some point when there is no possibility of an endovascular nor a surgical procedure, some alternative non-reconstructive options have been proposed such as lumbar sympathectomy and spinal cord stimulation. But their effectiveness is limited especially when assessing the results on objective criteria. The additional value of cell-based therapies has still to be proven from large RCTs and should therefore still be confined to a research setting. Altogether this chapter summarises the best available evidence for the treatment of CLI, which is, from multiple perspectives, completely different from claudication. The latter also stresses the importance of well-designed RCTs focusing on CLI patients reporting standardised endpoints, both clinical as well as procedural.

Chapter V: Diabetic Foot

Ulcerated diabetic foot is a complex problem. Ischaemia, neuropathy and infection are the three pathological components that lead to diabetic foot complications, and they frequently occur together as an aetiologic triad. Neuropathy and ischaemia are the initiating factors, most often together as neuroischaemia, whereas infection is mostly a consequence. The role of peripheral arterial disease in diabetic foot has long been underestimated as typical ischaemic symptoms are less frequent in diabetics with ischaemia than in non-diabetics. Furthermore, the healing of a neuroischaemic ulcer is hampered by microvascular dysfunction. Therefore, the threshold for revascularising neuroischaemic ulcers should be lower than that for purely ischaemic ulcers. Previous guidelines have largely ignored these specific demands related to ulcerated neuroischaemic diabetic feet. Any diabetic foot ulcer should always be considered to have vascular impairment unless otherwise proven. Early referral, non-invasive vascular testing, imaging and intervention are crucial to improve diabetic foot ulcer healing and to prevent amputation. Timing is essential, as the window of opportunity to heal the ulcer and save the leg is easily missed. This chapter underlines the paucity of data on the best way to diagnose and treat these diabetic patients. Most of the studies dealing with neuroischaemic diabetic feet are not comparable in terms of patient populations, interventions or outcome. Therefore, there is an urgent need for a paradigm shift in diabetic foot care; that is, a new approach and classification of diabetics with vascular impairment in regard to clinical practice and research. A multidisciplinary approach needs to implemented systematically with a vascular surgeon as an integrated member. New strategies must be developed and implemented for diabetic foot patients with vascular impairment, to improve healing, to speed up healing rate and to avoid amputation, irrespective of the intervention technology chosen. Focused studies on the value of predictive tests, new treatment modalities as well as selective and targeted strategies are needed. As specific data on ulcerated neuroischaemic diabetic feet are scarce, recommendations are often of low grade.

Below-the-knee angioplasty in patients with end-stage renal disease.

Purpose: To determine clinical efficacy of below-the-knee (BTK) angioplasty in patients with end-stage renal disease (ESRD). Methods: Interrogation of a prospectively maintained database containing 2659 patients treated at a tertiary referral hospital between February 1995 and June 2004 identified 29 ESRD patients (21 men; median age 69 years, IQR 10.12) who had 73 infrapopliteal atherosclerotic lesions treated in 38 ischemic limbs. The indication for treatment was intermittent claudication in 13 (34%) and critical limb ischemia in 25 (66%) limbs. BTK angioplasty was attempted either alone (n=18) or combined with an endovascular inflow procedure (n=20). Primary clinical success was defined as hemodynamic improvement (ABI increase ≥0.1) and/or symptomatic improvement (at least one clinical category). Cumulative rates were calculated according to the Kaplan-Meier estimate. Results: Primary technical success reached 97%, whereas hemodynamic improvement was obtained in only 50% (19/38) of the limbs treated. The pedal arteries were severely diseased in all, and complete occlusion of the pedal arch was found in 58% (18/31) of limbs on completion angiography. Median follow-up was 5.9 months (IQR 11.5). Primary clinical success was 17%, 11%, 11%, and 11% in patients with BTK angioplasty alone and 53%, 45%, 45%, and 45% in patients with inflow procedures after 3, 6, 9, and 12 months, respectively (p=0.017). Limb salvage was 73% at 12 months. Subgroup analyses showed significantly better clinical results in men (p=0.003) and in patients on hemodialysis compared to peritoneal dialysis (p=0.037). Conclusions: Clinical efficacy of BTK angioplasty is limited in patients with ESRD because of the severely diseased pedal arteries. Further studies are warranted to define subgroups of patients likely to experience a more favorable outcome.

Impact of obesity on venous hemodynamics of the lower limbs

BACKGROUND Obesity is a risk factor for chronic venous insufficiency and venous thromboembolism. The aim of this study was to compare venous flow parameters of the lower limbs assessed by duplex ultrasound scanning in obese and nonobese individuals according to body mass index (BMI). METHODS Venous hemodynamics were studied in a prospective cohort study in nonobese (BMI <25 kg/m(2)) and obese individuals (BMI >30 kg/m(2)). Diameter, flow volume, peak, mean, and minimum velocities were assessed. RESULTS The study examined 36 limbs in 23 nonobese individuals and 44 limbs in 22 obese individuals. The diameter of the femoral vein was significantly greater in obese (8.5 +/- 2.2 mm) vs nonobese (7.1 +/- 1.6 mm; P = .0009) limbs. Venous peak and minimum velocities differed between nonobese and obese individuals (14.8 +/- 7.2 vs 10.8 +/- 4.8 cm/s [P = .0071] and 4.0 +/- 3.6 vs 1.7 +/- 6.3 cm/s [P = .056]). Calculation of venous amplitude and shear stress showed significantly higher values in nonobese vs obese (18.8 +/- 9.4 vs 12.5 +/- 9.3 cm/s [P = .003] and 2.13 +/- 2.2 dyn/cm(2) vs 1.6 +/- 2.7 dyn/cm(2) [P = .03]). Spearman rank correlation revealed a significant inverse correlation between waist-to-hip ratios and waist circumference and venous peak velocity, mean velocity, velocities amplitude (peak velocity-minimum velocity), and shear stress. CONCLUSION Lower limb venous flow parameters differ significantly between healthy obese and nonobese individuals. These findings support the mechanical role of abdominal adipose tissue potentially leading to elevated risk for both venous thromboembolism and chronic venous insufficiency.

Falsely high ankle-brachial index predicts major amputation in critical limb ischemia.

Falsely high ankle-brachial index (ABI) values are associated with an adverse clinical outcome in diabetes mellitus. The aim of the present study was to verify whether such an association also exists in patients with chronic critical limb ischemia (CLI) with and without diabetes. A total of 229 patients (74 ± 11 years, 136 males, 244 limbs with CLI) were followed for 262 ± 136 days. Incompressibility of lower limb arteries (ABI > 1.3) was found in 45 patients, and was associated with diabetes mellitus (p = 0.01) and renal insufficiency (p = 0.035). Limbs with incompressible ankle arteries had a higher rate of major amputation (p = 0.002 by log-rank). This association was confirmed by multivariate Cox regression analysis (relative risk [RR] 2.67; 95% CI 1.27-5.64, p = 0.01). The relationship between ABI > 1.3 and amputation rate persisted after subjects with diabetes and renal insufficiency had been removed from the analysis (RR 3.85; 95% CI 1.25-11.79, p = 0.018). Dividing limbs with measurable ankle pressure according to tertiles of ABI, the group in the second tertile (0.323 ≤ ABI ≤ 0.469) had the lowest amputation rate (4/64, 6.2%), and a U-shaped association between the occurrence of major amputation and ABI was evident. No association was found between ABI and mortality. In conclusion, this study demonstrates that falsely high ABI is an independent predictor of major amputation in patients with CLI.

Percutaneous management of vascular complications in patients undergoing transcatheter aortic valve implantation

OBJECTIVES This study sought to investigate the feasibility and safety of percutaneous management of vascular complications after transcatheter aortic valve implantation (TAVI). BACKGROUND Vascular complications after TAVI are frequent and outcomes after percutaneous management of these adverse events not well established. METHODS Between August 2007 and July 2010, 149 patients underwent transfemoral TAVI using a percutaneous approach. We compared outcomes of patients undergoing percutaneous management of vascular complications with patients free from vascular complications and performed duplex ultrasonography, fluoroscopy, and multislice computed tomography during follow-up. RESULTS A total of 27 patients (18%) experienced vascular complications consisting of incomplete arteriotomy closure (n = 19, 70%), dissection (n = 3, 11%), arterial perforation (n = 3, 11%), arterial occlusion (n = 1, 4%), and pseudoaneurysm (n = 1, 4%). Percutaneous stent graft implantation was successful in 21 of 23 (91%) patients, whereas 2 patients were treated by manual compression, 2 patients underwent urgent surgery, and 2 patients required delayed surgery. Rates of major adverse cardiac events at 30 days were similar among patients undergoing percutaneous management of vascular complications and those without vascular complications (9% vs. 8%, p = 1.00). After a median follow-up of 10.9 months, imaging showed no evidence of hemodynamically significant stenosis (mean peak velocity ratio: 1.2 ± 0.4). Stent fractures were observed in 4 stents (22%, type I [6%], type II [16%]) and were clinically silent in all cases. CONCLUSIONS Vascular complications after TAVI can be treated percutaneously as a bailout procedure with a high rate of technical success, and clinical outcomes are comparable to patients without vascular complications. Stent patency is high during follow-up, although stent fractures require careful scrutiny.

Chapter II: Diagnostic Methods

Non-invasive vascular studies can provide crucial information on the presence, location, and severity of critical limb ischaemia (CLI), as well as the initial assessment or treatment planning. Ankle-brachial index with Doppler ultrasound, despite limitations in diabetic and end-stage renal failure patients, is the first-line evaluation of CLI. In this group of patients, toe-brachial index measurement may better establish the diagnosis. Other non-invasive measurements, such as segmental limb pressure, continuous-wave Doppler analysis and pulse volume recording, are of limited accuracy. Transcutaneous oxygen pressure (TcPO(2)) measurement may be of value when rest pain and ulcerations of the foot are present. Duplex ultrasound is the most important non-invasive tool in CLI patients combining haemodynamic evaluation with imaging modality. Computed tomography angiography (CTA) and magnetic resonance angiography (MRA) are the next imaging studies in the algorithm for CLI. Both CTA and MRA have been proven effective in aiding the decision-making of clinicians and accurate planning of intervention. The data acquired with CTA and MRA can be manipulated in a multiplanar and 3D fashion and can offer exquisite detail. CTA results are generally equivalent to MRA, and both compare favourably with contrast angiography. The individual use of different imaging modalities depends on local availability, experience, and costs. Contrast angiography represents the gold standard, provides detailed information about arterial anatomy, and is recommended when revascularisation is needed.