Predictors of bleeding complications during catHeter-dirEcted thrombolysis for peripheral arterial occlusions (POCHET).

INTRODUCTION: The risk of major bleeding complications in catheter directed thrombolysis (CDT) for acute limb ischemia (ALI) remains high, with reported major bleeding complication rates in up to 1 in every 10 treated patients. Fibrinogen was the only predictive marker used for bleeding complications in CDT, despite the lack of high quality evidence to support this. Therefore, recent international guidelines recommend against the use of fibrinogen during CDT. However, no alternative biomarkers exist to effectively predict CDT-related bleeding complications. The aim of the POCHET biobank is to prospectively assess the rate and etiology of bleeding complications during CDT and to provide a biobank of blood samples to investigate potential novel biomarkers to predict bleeding complications during CDT. METHODS: The POCHET biobank is a multicentre prospective biobank. After informed consent, all consecutive patients with lower extremity ALI eligible for CDT are included. All patients are treated according to a predefined standard operating procedure which is aligned in all participating centres. Baseline and follow-up data are collected. Prior to CDT and subsequently every six hours, venous blood samples are obtained and stored in the biobank for future analyses. The primary outcome is the occurrence of non-access related major bleeding complications, which is assessed by an independent adjudication committee. Secondary outcomes are non-major bleeding complications and other CDT related complications. Proposed biomarkers to be investigated include fibrinogen, to end the debate on its usefulness, anti-plasmin and D-Dimer. DISCUSSION AND CONCLUSION: The POCHET biobank provides contemporary data and outcomes of patients during CDT for ALI, coupled with their blood samples taken prior and during CDT. Thereby, the POCHET biobank is a real world monitor on biomarkers during CDT, supporting a broad spectrum of future research for the identification of patients at high risk for bleeding complications during CDT and to identify new biomarkers to enhance safety in CDT treatment.

Editor's Choice - Two Year Results of the Randomised DISCOVER Trial Comparing Covered Versus Bare Metal Stents in the Common Iliac Artery.

OBJECTIVE: It has been suggested that covered stents (CS) may lower restenosis rates compared with bare metal stents (BMS) after endovascular treatment of the common iliac artery. This trial aimed to provide additional evidence on the efficacy of CS vs. BMS in the common iliac artery. METHODS: This multicentre, randomised, single blind controlled superiority trial compared balloon expandable CS and balloon expandable BMS for advanced atherosclerotic lesions in the common iliac artery; this was defined as a stenosis > 3 cm in length or occlusion. The primary end point was freedom from binary restenosis after two years of follow up. The study was conducted according to the principles of the Declaration of Helsinki (version: October 2008) and registered with the Dutch Trial register (NTR3381). RESULTS: One hundred and seventy-four limbs were included between 2012 and 2019 with 87 limbs in each group. Six patients crossed over from the BMS group to the CS group but were analysed according to an intention to treat principle. Freedom from binary restenosis after two years of follow up was 84.7% (95% CI 76.7 - 92.7%) in the BMS group and 89.1% (95% CI 82.4 - 95.8%) in the CS group (p = .40). Freedom from occlusion was 95.0% (95% CI 90.3 - 95.7%) in the BMS group and 96.4% (95% CI 92.5 - 100%) in the CS group (p = .66). Freedom from target lesion revascularisation was 91.1% (95% CI 84.8 - 97.3%) and 95.2% (95% CI 90.7 -99.7%), respectively (p = .31). Technical success, complications, haemodynamic success, and clinical success were also comparable between both groups. Per-protocol analysis did not affect the outcomes of the study. CONCLUSION: No difference was found between balloon expandable CS and BMS for treating advanced atherosclerotic lesions of the common iliac artery.

Omniflow II biosynthetic grafts versus expanded polytetrafluoroethylene grafts for infrainguinal bypass surgery. A single-center retrospective analysis.

INTRODUCTION: The aim of this study is to evaluate the outcome of Omniflow II biosynthetic vascular grafts as compared to synthetic expanded polytetrafluoroethylene (ePTFE) grafts in infrainguinal bypass surgery. METHODS: A single-center, retrospective, observational study was performed reviewing patients with critical limb ischemia who underwent infrainguinal bypass surgery between 2014 and 2018. Patients characteristics, graft characteristics, and treatment outcomes were collected. Patency rates were compared using Kaplan-Meier estimates. RESULTS: Sixty bypasses were performed in 57 patients. For above-knee surgery, six were Omniflow and 13 were synthetic. For below-knee surgery, 19 were Omniflow and 22 were synthetic. Patient characteristics between groups were similar. However, American Society of Anesthesiologists (ASA) classification scores were higher in the Omniflow group as compared to ePTFE (88% was ASA 3 or higher versus 60%; p = 0.018). Furthermore, wound, ischemia, and foot infection (WIfI) composite scores were higher in the Omniflow group (p = 0.0001). There was a trend toward more active infection at time of surgery in the Omniflow group (40 vs 22.9%, p = 0.15). At 1 year, primary patency rates were 60.0% versus 46.9% for above-knee Omniflow versus ePTFE grafts, respectively (p = 0.72). Secondary patency rates were 80.0% versus 82.5% (p = 0.89), and limb salvage rates were 83.3% versus 100% (p = 0.14). For below-knee surgery, 1- and 2-year primary patency rates in Omniflow versus ePTFE grafts were 36.0% versus 41.8% (p = 0.60) and 36.0% versus 31.1% (p = 0.87). Secondary patency rates were 66.8% versus 75.2% at 1 year (p = 0.53) and 58.8% versus 48.3% (p = 0.77) at 2 years. Below-knee limb salvage rates for Omniflow versus ePTFE after 2 years were 88.0% versus 68.3% (p = 0.28), respectively. Aneurysmal degeneration occurred in 2/25 (8%) in the Omniflow group and 0/35 (0%) in the ePTFE group. Bypass infections occurred in 2/25 (8%) in the Omniflow group and 0/35 (0%) in the ePTFE group (p = 0.09). CONCLUSION: Omniflow bypasses were more commonly implanted in patients with higher limb infection rate as confirmed with a higher adapted WIfI score. A trend toward a higher infection rate of Omniflow grafts was observed but not statistically significant. Graft infection rates were relatively low and treatable with antibiotics. No significant difference in graft performance was observed. The choice between the two studied grafts remains based on surgeon's preference.

Clinical validation of 2D perfusion angiography using Syngo iFlow software during peripheral arterial interventions.

OBJECTIVE: Endovascular surgery is an important treatment modality in peripheral arterial disease. Digital subtraction angiography is the standard post revascularisation diagnostic tool to locate lesions and to evaluate the effect of an intervention. However, interpretation of digital subtraction angiography images is subjective and it is difficult to determine whether revascularisation has been sufficient for clinical improvement. A new technique is 2D perfusion angiography, which creates a 2D colour map and time density curve from the digital subtraction angiography scan for an objective evaluation of the results. However, its clinical relevance is unknown. The aim is to evaluate the association between 2D perfusion angiography parameters and clinical outcome after peripheral arterial interventions. METHODS: In this retrospective study, post revascularisation angiographic data and clinical data were reviewed of patients who underwent treatment of femoral-popliteal or femoral-tibial arteries. The outcome was assessed at three time points using three classification systems for peripheral arterial disease: Fontaine classification, American Medical Association whole person impairment classification (AMA) and average wound, ischemia, foot infection score. Post revascularisation angiographic data consisted of time density curves of the foot and lower leg which were extracted from the Syngo iFlow system (Siemens Healthineers). For each time density curve, five descriptive parameters were calculated: time of arrival, time to peak, mean transit time, wash-in rate and area under the curve. The association between the time density curve parameters and peripheral arterial disease classification systems was assessed using a regression analysis. RESULTS: Between July 2016 and December 2018, 103 patients underwent peripheral endovascular interventions in the hybrid operating room; 39 patients were suitable for analysis, of which 28 patients underwent digital subtraction angiography of the lower leg, 3 patients underwent digital subtraction angiography of the foot and 8 patients underwent digital subtraction angiography of both regions. Limited significant relations were found for time of arrival with Fontainde classification (B = 0.806, p = 0.043) and area under the curve with AMA classification (B = -0.027, p = 0.047). CONCLUSION: In this retrospective study, time density curve parameters (time of arrival and area under the curve), measured in the lower leg, showed a limited significant association with two classification systems for peripheral arterial disease. Future prospective studies to determine the clinical relevance of this 2D perfusion angiography method should focus on standardisation of angiography protocols and comparison of pre- and post-intervention parameters.

Angioplasty versus stenting for iliac artery lesions.

BACKGROUND: Atherosclerosis of the iliac artery may result in a stenosis or occlusion, which is defined as iliac artery occlusive disease. A range of surgical and endovascular treatment options are available. Open surgical procedures have excellent patency rates but at the cost of substantial morbidity and mortality. Endovascular treatment has good safety and short-term efficacy with decreased morbidity, complications and costs compared with open surgical procedures. Both percutaneous transluminal angioplasty (PTA) and stenting are commonly used endovascular treatment options for iliac artery occlusive disease. A stenotic or occlusive lesion of the iliac artery can be treated successfully by PTA alone. If PTA alone is technically unsuccessful, additional stent placement is indicated. Alternatively, a stent could be placed primarily to treat an iliac artery stenosis or occlusion (primary stenting, PS). However, there is limited evidence to prove which endovascular treatment strategy is superior for stenotic and occlusive lesions of the iliac arteries. This is an update of the review first published in 2015. OBJECTIVES: To assess the effects of percutaneous transluminal angioplasty versus primary stenting for stenotic and occlusive lesions of the iliac artery. SEARCH METHODS: The Cochrane Vascular Information Specialist searched the Cochrane Vascular Specialised Register, CENTRAL, MEDLINE, Embase and CINAHL databases and World Health Organization International Clinical Trials Registry Platform and ClinicalTrials.gov trials registers to 24 September 2019. We also undertook reference checking and citation searching to identify additional studies. SELECTION CRITERIA: We included all randomised controlled trials (RCTs) comparing percutaneous transluminal angioplasty and primary stenting for iliac artery occlusive disease. We excluded quasi-randomised trials, case reports, case-control or cohort studies. We did not exclude studies based on the language of publication. DATA COLLECTION AND ANALYSIS: Two authors independently selected suitable trials, extracted data, assessed trial quality and performed data analyses. When there was disagreement, consensus would be reached first by discussion between the two authors and, if needed, through consultation with a third author. We used GRADE criteria to assess the certainty of the evidence and presented the main results in a 'Summary of findings' table. The main outcomes of interest were technical success, complications, symptomatic improvement of peripheral arterial disease (PAD), patency, reinterventions, resolutions of symptoms and signs, and improvement in walking distance as reported by the patient. MAIN RESULTS: We identified no new studies for this update. Previously, we identified two RCTs, with a combined total of 397 participants, as meeting the selection criteria. One study included mostly stenotic lesions (95%), whereas the second study included only iliac artery occlusions. Heterogeneity between these two studies meant it was not possible to pool the data. Both studies were of moderate methodological quality with some risk of bias relating to selective reporting and non-blinding of participants and personnel. Both studies occurred in the 1990s and techniques have since evolved. We assessed the overall certainty of the evidence to be low. We downgraded by two levels: one for risk of bias concerns and one for imprecision and indirectness. There was no evidence of a difference following percutaneous transluminal angioplasty (PTA) with selective stenting compared to primary stenting (PS) in technical success rates in either the study involving stenotic lesions (odds ratio (OR) 1.51, 95% confidence interval (CI) 0.77 to 2.99; 279 participants; low certainty evidence); or the study involving iliac artery occlusions (OR 2.95, 95% CI 0.12 to 73.90; 112 participants; low certainty evidence). In one trial, PTA of iliac artery occlusions resulted in a higher rate of major complications, especially distal embolisation (OR 4.50 95% CI 1.18 to 17.14; 1 study, 112 participants; low certainty evidence). Immediate complications were similar in the second study (OR 1.81, 95% CI 0.64 to 5.13; 1 study, 279 participants; low certainty evidence). Neither study reported on delayed complications. No evidence of a difference was seen in symptomatic improvement (OR 1.03, 95% CI 0.47 to 2.27; 1 study, 157 participants; low certainty evidence). The second study did not provide data but reported no differences. For the outcome of patency, no evidence of a difference was seen in the study involving iliac occlusion at two years (OR 1.60, 95% CI 0.34 to 7.44; 1 study, 57 participants; low certainty evidence); or the study involving stenotic lesions at two years (71.3% in the PS group versus 69.9% in the PTA group). Only one study reported on reintervention (six to eight years, OR 1.22, 95% CI 0.67 to 2.23; 1 study, 279 participants; low certainty evidence); and resolution of symptoms and signs (12 months, OR 1.14, 95% CI 0.65 to 2.00; 1 study, 219 participants; low certainty evidence), with no evidence of a difference detected in either outcome. Neither study reported on improvement in walking distance as reported by the patient. AUTHORS' CONCLUSIONS: There is insufficient evidence to make general conclusions about the effects of percutaneous transluminal angioplasty versus primary stenting for stenotic and occlusive lesions of the iliac artery. Data from one study indicate that primary stenting in iliac artery occlusions may result in lower distal embolisation rates (low certainty evidence). The evidence in this review, based on two studies, was assessed as low certainty, with downgrading decisions based on limitations in risk of bias, imprecision and indirectness. More studies are required to strengthen our confidence in the results.

Angioplasty versus stenting for iliac artery lesions.

BACKGROUND: Atherosclerosis of the iliac artery may result in a stenosis or occlusion, which is defined as iliac artery occlusive disease. A range of surgical and endovascular treatment options are available. Open surgical procedures have excellent patency rates but at the cost of substantial morbidity and mortality. Endovascular treatment has good safety and short-term efficacy with decreased morbidity, complications and costs compared with open surgical procedures. Both percutaneous transluminal angioplasty (PTA) and stenting are commonly used endovascular treatment options for iliac artery occlusive disease. A stenotic or occlusive lesion of the iliac artery can be treated successfully by PTA alone. If PTA alone is technically unsuccessful, additional stent placement is indicated. Alternatively, a stent could be placed primarily to treat an iliac artery stenosis or occlusion (primary stenting, PS). However, there is limited evidence to prove which endovascular treatment strategy is superior for stenotic and occlusive lesions of the iliac arteries. OBJECTIVES: To assess the effects of percutaneous transluminal angioplasty versus primary stenting for stenotic and occlusive lesions of the iliac artery. SEARCH METHODS: The Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched April 2015) and Cochrane Register of Studies (CRS) (2015, Issue 3). The TSC searched trial databases for details of ongoing and unpublished studies. SELECTION CRITERIA: We included all randomised controlled trials (RCTs) comparing percutaneous transluminal angioplasty and primary stenting for iliac artery occlusive disease. We excluded quasi-randomised trials, case reports, case-control or cohort studies. We excluded no studies based on the language of publication. DATA COLLECTION AND ANALYSIS: Two authors (JB, NA) independently selected suitable trials. JB and HJ independently performed data extraction and trial quality assessment. When there was disagreement, consensus would be reached first by discussion among both authors and, if still no consensus could be reached, through consultation with BF. MAIN RESULTS: We identified two RCTs with a combined total of 397 participants as meeting the selection criteria. One study included mostly stenotic lesions (95%), whereas the second study included only iliac artery occlusions. Both studies were of moderate methodological quality with some risk of bias relating to selective reporting and non-blinding of participants and personnel. The overall quality of evidence was low due to the small number of included studies, the differences in study populations and definitions of the outcome variables. Due to the heterogeneity among these two studies it was not possible to pool the data. Percutaneous transluminal angioplasty (PTA) with selective stenting and primary stenting (PS) resulted in similar improvement in the stage of peripheral arterial occlusive disease according to Rutherford's criteria, resolution of symptoms and signs, improvement of quality of life, technical success of the procedure and patency of the treated vessel. Improvement in walking distance as reported by the patient, measured claudication distance, ulcer healing, major amputation-free survival and delayed complications (> 72 hours) were not reported in either of the studies. In one trial, PTA of iliac artery occlusions resulted in a significantly higher rate of major complications, especially distal embolisation. The other trial showed a significantly higher mean ankle brachial index (ABI) at two years in the PTA group (1.0) compared to the mean ABI in the PS group (0.91); mean difference (MD) 0.09 (95% confidence interval (CI) 0.04 to 0.14; P value = 0.001, analysis performed by review authors). However, at other time points there was no difference. We consider it unlikely that this difference is attributable to the study procedure, and also believe this difference may not be clinically relevant. AUTHORS' CONCLUSIONS: There is insufficient evidence to assess the effects of PTA versus PS for stenotic and occlusive lesions of the iliac artery. From one study it appears that PS in iliac artery occlusions may result in lower distal embolisation rates. More studies are required to come to a firm conclusion.

Ruptured abdominal aortic aneurysm in a patient with an aorto-left renal vein fistula.

The combination of an aorto-left renal vein fistula (ALRVF) and a ruptured abdominal aortic aneurysm is extremely rare, with only 9 cases having been reported. We present such a case and discuss the previously reported cases and treatment strategies.

[Chewing gum to prevent postoperative ileus]. / Kauwgom kauwen ter preventie van postoperatieve ileus.

Postoperative ileus is a commonly occurring complication after abdominal surgery. Reduced well-being and ileus related complications lead to extension of hospital stay. An early commencement of postoperative feeding to stimulate the digestive system is not always achievable in practice. Recent studies suggest that use of chewing gum can be effective in preventing postoperative ileus by a similar mechanism of action to early postoperative feeding. However, these studies were small in size and of varying quality. Recently the "Chewing gum study" ("Kauwgomstudie") to investigate the effect of general use of chewing gum after abdominal surgery has been started in the Netherlands.

[A woman with acute upper abdominal pain]. / Een vrouw met acute pijn in epigastrio.

A 42-year-old woman came to the emergency department with acute abdominal pain. CT-scan showed an isolated dissection of the A. mesenterica superior and signs of ischaemic colitis. The patient was treated with endovascular stent placement in the artery and partial small bowel resection.

Critical role of tissue kallikrein in vessel formation and maturation: implications for therapeutic revascularization.

OBJECTIVE: Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function polymorphic variant, rs5515 (R53H), which possesses reduced kinin-forming activity. METHODS AND RESULTS: Here, we demonstrate that tissue kallikrein knockout mice (KLK1-/-) show impaired muscle neovascularization in response to hindlimb ischemia. Gene-transfer of wild-type Ad.hKLK1 but not Ad.R53H-hKLK1 was able to rescue this defect. Similarly, in the rat mesenteric assay, Ad.hKLK1 induced a mature neovasculature with increased vessel diameter through kinin-B2 receptor-mediated recruitment of pericytes and vascular smooth muscle cells, whereas Ad.R53H-hKLK1 was ineffective. Moreover, hKLK1 but not R53H-hKLK1 overexpression in the zebrafish induced endothelial precursor cell migration and vascular remodeling. Furthermore, Ad.hKLK1 activates metalloproteinase (MMP) activity in normoperfused muscle and fails to promote reparative neovascularization in ischemic MMP9-/- mice, whereas its proarteriogenic action was preserved in ApoE-/- mice, an atherosclerotic model of impaired angiogenesis. CONCLUSIONS: These results demonstrate the fundamental role of endogenous Tissue Kallikrein in vascular repair and provide novel information on the cellular and molecular mechanisms responsible for the robust arterialization induced by hKLK1 overexpression.

Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.

For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a plasmid in which two opposing human polymerase III promoters (H1 and U6) drive the expression of siRNA constructs that form functional double-stranded siRNAs, in combination with in vivo bioluminescence imaging, we were able to knock down exogenous delivered luciferase for at least 100 days in murine calf muscles. This effect was sequence specific, because scrambled siRNA had no effect. Moreover, we were able to demonstrate in vivo reduction of endogenous TLR4 expression for at least 1 week, using a similar vector expressing an siRNA for TLR4 in the muscle. In this study, we demonstrate that in vivo suppression of both endogenous (for at least 1 week) and introduced genes (>100 days) is feasible via plasmid-driven siRNA expression after electroporation-mediated intramuscular gene transfer. With this approach the short-term effect of oligonucleotides and the drawbacks of viral gene delivery, like immunological responses, could be circumvented. Therefore, this application of RNA interference is a useful tool with which to investigate gene function and might be promising as a therapeutic tool for locally acting diseases such as restenosis or tumors.

Vaccination against VEGFR2 attenuates initiation and progression of atherosclerosis.

OBJECTIVE: Vascular endothelial growth factor receptor 2 (VEGFR2)-overexpressing cells may form an interesting target for the treatment of atherosclerosis because of their involvement in processes that contribute to this disease, such as angiogenesis. METHODS AND RESULTS: We vaccinated mice against VEGFR2 by an orally administered DNA vaccine, comprising a plasmid, encoding murine VEGFR2, carried by live attenuated Salmonella typhimurium. This vaccine induces cellular immunity against cells that overexpress VEGFR2. Vaccination of hypercholesterolemic mice against VEGFR2 resulted in a marked induction of CD8+ cytotoxic T cells specific for VEGFR2 and led to an inhibition of angiogenesis in a hindlimb ischemia model. Interestingly, VEGFR2 vaccination attenuated the progression of preexisting advanced atherosclerotic lesions in the brachiocephalic artery of apoE-/- mice. Furthermore, VEGFR2 vaccination strongly reduced the initiation of collar-induced atherosclerosis in the carotid arteries of LDLr-/- mice. In addition, denudation of the carotid artery, as a model for postinterventional lesion formation, resulted in delayed endothelial replacement and significantly increased neointima formation on VEGFR2 vaccination. CONCLUSIONS: These data indicate the prominent role of VEGFR2+ cells in cardiovascular diseases and show that induction of cellular immunity against atherosclerosis-associated cells by means of DNA vaccination may contribute to the development of novel therapies against atherosclerosis.

Expression of vascular endothelial growth factor, stromal cell-derived factor-1, and CXCR4 in human limb muscle with acute and chronic ischemia.

OBJECTIVE: Vascular endothelial growth factor (VEGF)-induced stromal cell-derived factor-1 (SDF-1) has been implicated in angiogenesis in ischemic tissues by recruitment of CXCR4-positive bone marrow-derived circulating cells with paracrine functions in preclinical models. Here, evidence for this is provided in patients with peripheral artery disease. METHODS AND RESULTS: Expression patterns of VEGF, SDF-1, and CXCR4 were studied in amputated limbs of 16 patients. VEGF-A was expressed in vascular structures and myofibers. SDF-1 was expressed in endothelial and subendothelial cells, whereas CXCR4 was expressed in proximity to capillaries. VEGF-A, SDF-1, and CXCR4 expressions were generally decreased in ischemic muscle as compared with nonischemic muscle in patients with chronic ischemia (0.41-fold, 0.97-fold, and 0.54-fold induction [medians], respectively), whereas substantially increased in 2 patients with acute-on-chronic ischemia (3.5- to 65.8-fold, 3.9- to 19.0-fold, and 4.1- to 30.6-fold induction, respectively). Furthermore, these gene expressions strongly correlated with capillary area. Only acute ischemic tissue displayed a high percentage of hypoxia-inducible factor-1alpha-positive nuclei. CONCLUSIONS: These data suggest that VEGF and SDF-1 function as pro-angiogenic factors in patients with ischemic disease by perivascular retention of CXCR4-positive cells. Furthermore, these genes are downregulated in chronic ischemia as opposed to upregulated in more acute ischemia. The VEGF-SDF-1-CXCR4 pathway is a promising target to treat chronic ischemic disease.

Hypercholesterolemia reduces collateral artery growth more dominantly than hyperglycemia or insulin resistance in mice.

OBJECTIVE: Collateral artery development (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease, may be deregulated by vascular risk factors, eg, diabetes or hypercholesterolemia. Here, we compared the effects of either disturbed glucose metabolism or disturbed lipid metabolism on arteriogenesis. METHODS AND RESULTS: Femoral artery occlusion was performed in streptozotocin(STZ)-treated mice, nonobese diabetic (NOD) mice, and insulin-resistant Ob/Ob mice on regular diet, and APOE3*Leiden mice on different hypercholesterolemic diets. Angiography and laser Doppler perfusion analysis of hindlimbs were performed postoperatively. Surprisingly, angiographic arteriogenesis was not impaired in diabetic and insulin-resistant mice. Perfusion recovery in STZ-treated and Ob/Ob mice was only decreased by 19% and 16%, respectively (P<0.05). Furthermore, perfusion recovery was unchanged between high-glycemic and mild-glycemic NOD mice. Angiographic arteriogenesis in APOE3*Leiden mice, however, was markedly impaired at 7 days and 14 days (P< or =0.01). Correspondingly, perfusion recovery was 41% decreased in APOE3*Leiden mice (P<0.05). There was an inverse correlation of perfusion recovery with plasma cholesterol (P=0.02), but not with triglyceride, free fatty acid, glucose, or insulin levels. CONCLUSIONS: Hypercholesterolemia reduces arteriogenesis more dominantly than hyperglycemia or hyperinsulinemia in mice. This suggests that a disturbed lipid metabolism as observed in diabetic patients might be crucial for the impairment of collateral formation.

Vascular endothelial growth factor overexpression in ischemic skeletal muscle enhances myoglobin expression in vivo.

Therapeutic angiogenesis using vascular endothelial growth factor (VEGF) is considered a promising new therapy for patients with arterial obstructive disease. Clinical improvements observed consist of improved muscle function and regression of rest pain or angina. However, direct evidence for improved vascularization, as evaluated by angiography, is weak. In this study, we report an angiogenesis-independent effect of VEGF on ischemic skeletal muscle, ie, upregulation of myoglobin after VEGF treatment. Mice received intramuscular injection with adenoviral VEGF-A or either adenoviral LacZ or PBS as control, followed by surgical induction of acute hindlimb ischemia at day 3. At day 6, capillary density was increased in calf muscle of Ad.VEGF-treated versus control mice (P<0.01). However, angiographic score of collateral arteries was unchanged between Ad.VEGF-treated and control mice. More interestingly, an increase in myoglobin was observed in Ad.VEGF-treated mice. Active myoglobin was 1.5-fold increased in calf muscle of Ad.VEGF-treated mice (P< or =0.01). In addition, the number of myoglobin-stained myofibers was 2.6-fold increased in Ad.VEGF-treated mice (P=0.001). Furthermore, in ischemic muscle of 15 limb amputation patients, VEGF and myoglobin were coexpressed. Finally, in cultured C2C12 myotubes treated with rhVEGF, myoglobin mRNA was 2.8-fold raised as compared with PBS-treated cells (P=0.02). This effect could be blocked with the VEGF receptor tyrosine kinase inhibitor SU5416. In conclusion, we show that VEGF upregulates myoglobin in ischemic muscle both in vitro and in vivo. Increased myoglobin expression in VEGF-treated muscle implies an improved muscle oxygenation, which may, at least partly, explain observed clinical improvements in VEGF-treated patients, in the absence of improved vascularization.