Bright quantum dots emitting at ∼1,600 nm in the NIR-IIb window for deep tissue fluorescence imaging.

With suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in the 1,500- to 1,700-nm range of the near-IR (NIR) spectrum (NIR-IIb window) can afford high clarity and deep tissue penetration. However, there has been a lack of NIR-IIb fluorescent probes with sufficient brightness and aqueous stability. Here, we present a bright fluorescent probe emitting at ∼1,600 nm based on core/shell lead sulfide/cadmium sulfide (CdS) quantum dots (CSQDs) synthesized in organic phase. The CdS shell plays a critical role of protecting the lead sulfide (PbS) core from oxidation and retaining its bright fluorescence through the process of amphiphilic polymer coating and transferring to water needed for imparting aqueous stability and compatibility. The resulting CSQDs with a branched PEG outer layer exhibited a long blood circulation half-life of 7 hours and enabled through-skin, real-time imaging of blood flows in mouse vasculatures at an unprecedented 60 frames per second (fps) speed by detecting ∼1,600-nm fluorescence under 808-nm excitation. It also allowed through-skin in vivo confocal 3D imaging of tumor vasculatures in mice with an imaging depth of ∼1.2 mm. The PEG-CSQDs accumulated in tumor effectively through the enhanced permeation and retention effect, affording a high tumor-to-normal tissue ratio up to ∼32 owing to the bright ∼1,600-nm emission and nearly zero autofluorescence background resulting from a large ∼800-nm Stoke's shift. The aqueous-compatible CSQDs are excreted through the biliary pathway without causing obvious toxicity effects, suggesting a useful class of ∼1,600-nm emitting probes for biomedical research.

Contemporary management of critical lower limb ischemia in TASC D lesions with subintimal angioplasty in femoro-popliteal lesions, tibial angioplasty and sequential compression biomechanical device for infra-inguinal arterial occlusion. Experience and quality of life outcome learned over 25 years.

AIM: Patients with end-stage critical limb ischemia (CLI) survive on borrowed time and amputation is inevitable if an aggressive management stratagem is not instigated. Our primary aim was to equate effectiveness of subintimal angioplasty (SIA) and tibial balloon angioplasty (TBA) in sustaining clinical improvement and amputation free survival (AFS) in patients with CLI TASD II D. Moreover, patients with severe CLI, who were not suitable for revascularization and who were offered therapy with a sequential compression biomechanical device (SCBD) were scrutinised as part of a comprehensive lower limb salvage program. METHODS: From 2002-2012, 5876 patients were referred with peripheral vascular disease (PVD); 987 presented with CLI and 798 had intervention; 189 patients presenting with CLI were not candidates for revascularisation, out of which 171 were offered SCBD. We formed a prospective observational group study of 441 patient who had TASC D disease. All of these patients presented as emergencies and were allocated to the next available treatment list. Duplex ultrasound arterial mapping (DUAM) was the sole preoperative investigation tool in 92% of all cases. Of the 441 patients studied, 190 patients (206 procedures) has SIA for TASC D femero-popliteal occlusions, 80 patients (89 procedures) had TBA and cool eximer laser angioplasty (CELA) for tibial artery occlusions and 171 patients with severe CLI were not suitable for revascularization and joined the SCBD program. Mean age (SIA 73±13 years vs. TBA/CELA 74±8 years vs. SCBD 75±13 years), and comorbidity severity scores (P>0.05) were similar between groups. RESULTS: Perioperative mortality within the SIA group was 1.6% vs. 0% within the TBA group and 0.6% in SCBD. Length of hospital stay within the TBA group was 3.8±2 days vs. SIA 14±16 days, P<0.0001. The 5-year freedom from major adverse events (MAE) for the SIA group was 68% that was comparable to the results obtained for both the TBA group; 59%, and SCBD group: 62.5% (P=0.1935). Five-year freedom from target lesion revascularization was 85.9% within the SIA group and 79% within the TBA group. A sustained clinical improvement was seen in 82.8% of primary SIA and 68% of TBA, which mimics the outcome of SCBD at 68% at one year. A total of 83% SCBD patients had no rest pain within one week of starting the program and gangrene remained dry and non-progressive. Ulceration healed in all but 12 patients. There were no device-related complications. Limb salvage was 94% at 5 years. All-cause survival was 69%. Quality time spent without symptoms of disease or toxicity of treatment (Q-TWiST) was 24.7 months for SIA and 8.5 months for TBA and was 38.13 for SCBD for a total of 708 months of usage. Cost per quality adjusted-life years (QALY) for SIA was € 5662.79, € 12,935.18 for TBA and € 2943.56 for SCBD. CONCLUSION: All treatment pathways augmented patient-specific Q-TWiST with substantial cost reduction. SIA, TBA and SCBD expand AFS and symptom-free survival. All treatment modalities are minimally invasive and allow for a high patient turnover without compromising limb salvage, once they are performed by experienced vascular surgeons in high deliberate practice volume centers.

Nonsuture anastomosis of arteries and veins using the magnetic pinned-ring device: a histologic and scanning electron microscopic study.

BACKGROUND: The goal of this study was to evaluate the performance of the magnetic pinned-ring device for nonsuture vascular anastomosis. METHODS: The magnetic pinned-ring device consists of paired magnetic rings that are coated with titanium nitride and embedded in a polypropylene shell; the rings are equipped with alternately spaced holes and titanium pins. The vascular anastomosis procedure using the novel magnetic pinned-ring device was performed on 14 mongrel dogs, and the traditional hand-sewing technique was used on 14 additional dogs. In situ end-to-end anastomoses were performed in the femoral artery and the inferior vena cava. Patency was confirmed through ultrasonographic scans at different time points as late as 24 weeks after surgery. Gross observation, histological staining, and scanning electron microscopy were used to evaluate the results at 24 weeks postoperatively. RESULTS: The time required to perform the vascular anastomosis was significantly shorter for the magnetic device than for hand sewing. A continuity of re-endothelialization was confirmed in all anastomotic stomas after 24 weeks, and neither formation of aneurysms nor thickening of the vascular wall was noted. The re-endothelialization was smooth at the anastomotic site of the magnetic device, whereas hand sewing resulted in rough and uneven re-endothelialization and the presence of visible sutures. Moreover, the endothelial cells were regularly arranged at the anastomotic site of the magnetic device, whereas different-sized and irregularly aligned endothelial cells were present at the hand-sewn anastomotic site. Use of the magnetic device was associated with significantly decreased deposition of fibrotic collagen and depressed infiltration of inflammatory cells compared with use of the hand-sewing technique. CONCLUSIONS: The magnetic pinned-ring device offers a simple, fast, reliable, and efficacious technique for nonsuture vascular anastomosis. Use of this device shortens operation time, maintains a high patency rate, and improves the healing of vascular tissue.

Knitted nitinol represents a new generation of constrictive external vein graft meshes.

OBJECTIVE: Constriction of vein grafts with braided external nitinol meshes had previously led to the successful elimination of neointimal tissue formation. We investigated whether pulse compliance, smaller kink-free bending radius, and milder medial atrophy can be achieved by knitting the meshes rather than braiding, without losing the suppressive effect on intimal hyperplasia. METHODS: Pulse compliance, bending stiffness, and bending radius, as well as longitudinal-radial deformation-coupling and radial compression, were compared in braided and knitted nitinol meshes. Identical to previous studies with braided mesh grafts, a senescent nonhuman primate model (Chacma baboons; bilateral femoral interposition grafts/6 months) mimicking the clinical size mismatch between vein grafts and runoff arteries was used to examine the effect of knitted external meshes on vein grafts: nitinol mesh-constricted (group 1); nitinol mesh-constricted and fibrin sealant (FS) spray-coated for mesh attachment (group 2); untreated control veins (group 3), and FS spray-coated control veins (group 4). RESULTS: Compared with braided meshes, knitted meshes had 3.8-times higher pulse compliance (3.43 ± 0.53 vs 0.94 ± 0.12%/100 mm Hg; P = .00002); 30-times lower bending stiffness (0.015 ± 0.002 vs 0.462 ± 0.077 Nmm(2); P = .0006); 9.2-times narrower kink-free bending radius (15.3 ± 0.4 vs 140.8 ± 22.4 mm; P = .0006), and 4.3-times lower radial narrowing caused by axial distension (18.0% ± 1.0% vs 77.0% ± 3.7%; P = .00001). Compared with mesh-supported grafts, neointimal tissue was 8.5-times thicker in group I (195 ± 45 µm) vs group III (23.0 ± 21.0 µm; P < .001) corresponding with a 14.3-times larger neointimal area in group I (4330 ± 957 × 103 µm(2)) vs group III (303 ± 221× 103 µm(2); P < .00004). FS had no significant influence. Medial muscle mass remained at 43.4% in knitted meshes vs the 28.1% previously observed in braided meshes. CONCLUSION: Combining the suppression of intimal hyperplasia with a more physiologic remodeling process of the media, manifold higher kink-resistance, and lower fraying than in braided meshes makes knitted nitinol an attractive concept in external vein graft protection.

[125Iodine-induced radiation damage in the femoral arteries of rabbits].

BACKGROUND & OBJECTIVE: What damages 125Iodine (125I) seed implantation will do to normal tissue is an observation topic in clinic. This study was to investigate the impact of 125I seed implantation to the femoral arteries of rabbits. METHODS: A total of 30 healthy rabbits were divided into 3 groups. The femoral sheath in the hind leg at one side selected by random was opened and 10 125I seeds at 0.79-0.85 mCi were implanted near the femoral artery according to the treatment planning system (TPS) before operation. In control group, 10 seeds without radioactivity were implanted. After 2 weeks, 2 months and 4 months, the rabbits were killed, the vessel wall nearest to the seeds was taken out and its morphologic changes were observed by gross examination, light microscopy and electron microscopy. RESULTS: In gross observation, the vessel segments of the legs with 125I seed implantation had no apparent abnormality. Under light microscope, a few exfoliative endothelial cells were observed. Under electron microscope, exfoliation of endothelial cells, degeneration of endothelial cells and smooth muscle cells were observed. The ratios of degenerative vessel endothelial cells to all vessel endothelial cells were 60%-70% in 2-week group, 50% in 2-month group and 30% in 4-month group; the ratios of degenerative vessel smooth muscle cells to all vessel smooth muscle cells in the 3 groups were 50%, 30% and 10%. CONCLUSION: 125I permanent implantation at 0.79-0.85 mCi mainly damages endothelial cells and smooth muscle cells of the normal vessels and this damnification is reversible.

Percutaneous transplantation of genetically-modified autologous fibroblasts in the rabbit femoral artery: A novel approach for cardiovascular gene therapy.

OBJECTIVE: Arterial cell and gene therapies are promising strategies for the treatment of cardiovascular diseases; however, the optimal cell type and delivery technique for such treatment remain to be determined. The aim of the present study was to design a new approach for arterial cell and gene therapy in which genetically modified autologous skin fibroblasts are percutaneously delivered in stented rabbit femoral arteries in vivo. METHODS: Autologous skin fibroblasts underwent in vitro transfection with the cationic lipid FuGene and plasmids expressing the human form of the tissue inhibitor of metalloproteinase (hTIMP-1) or nls-LacZ reporter genes. RESULT: Transfection efficiency was about 50% and there were high levels of hTIMP-1 secretion up to 14 days after gene transfer. We demonstrated the feasibility of in vivo percutaneous transplantation of fluorescent fibroblasts in the rabbit femoral artery. Results were confirmed by scanning electron microscopy. In vivo local delivery of hTIMP-1-expressing fibroblasts in stented femoral arteries also resulted in high-levels of hTIMP-1 secretion ex vivo for 7 days. Fibroblast transplantation resulted in a modest increase in intimal hyperplasia at the target site, which was reversed with hTIMP-1-transfected fibroblasts. CONCLUSION: Percutaneous transplantation of genetically modified autologous fibroblasts could be used as a cellular platform for locoregional secretion of therapeutic proteins to treat either specific arterial diseases or the diseased organ (eg, the heart) supplied by the target artery. CLINICAL RELEVANCE: Cell and gene therapies are potential new treatments for cardiovascular diseases. We demonstrated that autologous fibroblasts could be easily harvested from a skin biopsy specimen, genetically modified in vitro with nonviral vectors, and percutaneously seeded in vivo in rabbit femoral arteries, leading to locoregional secretion of abundant amounts of recombinant proteins. This new approach has important advantages over alternative approaches that use endothelial cells, viral vectors, and intraoperative cell delivery. Clinical applications may include local treatment of atherosclerotic plaques or aneurysms and also treatment of the diseased organs supplied by the target artery (eg, ischemic or failing heart).

The oestrogen receptor beta contributes to sex related differences in endothelial function of murine small arteries via EDHF.

Sex related differences in cardiovascular function have been reported in oestrogen receptor beta knockout (ERbetaKO) mice. In this study we examined the role of endothelium-derived hyperpolarizing factor (EDHF) in differences in small artery endothelial function between ERbetaKO and wild-type (WT) mice. Small femoral arteries were isolated from ERbetaKO and WT mice and mounted on a wire myograph. Concentration-response curves to ACh were compared before and after incubation with inhibitors of nitric oxide (NO) and prostacyclin (PGI2) synthesis. Comparison of the expression of the principal vascular connexins (Cx37, 40 and 43), implicated in EDHF-mediated dilatation were undertaken by immunohistochemistry. Vascular ultrastructure was studied by transmission and scanning electron microscopy. ACh-induced relaxation of arteries (< 200 microm internal diameter) was greater in WT females versus males and was attributable to a greater EDHF component of relaxation. This sex difference was absent in ERbetaKO mice. Arteries from ERbetaKO males (but not females) were more sensitive to ACh compared to WT. The pharmacological evidence and morphological prerequisite for involvement of gap junctions in EDHF-mediated responses was confirmed in male arteries. The absence of ERbeta had no influence on expression of main Cx subtypes within vascular wall or on ultrastructure and morphology of the endothelium. The data suggest that in WT male mice, ERbeta reduces EDHF-mediated relaxation through gap junction communication.

Short-term patency of 1 mm diameter PTFE prosthesis: an angiographic, Doppler-flowmetric and morphological experimental study.

Expanded polytetrafluoroethylene (PTFE) prostheses may be used in peripheral vascular surgery. Due to contradictory results on patency rate and neointimal formation, the effectiveness of this kind of prosthesis in small caliber artery reconstruction is still under discussion. In order to evaluate the effectiveness of this technique 1 mm internal diameter PTFE prosthesis (fibril length 35 microm) interposed in rabbit femoral artery, for 15 and 30 days, were studied functionally and morphologically. Doppler was performed during surgery, at day 2, 15 and 30. Arteriography was carried out at day 1. Light microscopy and scanning electron microscopy were performed on prosthesis sampled at day 15 and 30. Doppler flowmetry showed the full patency in all PTFE prosthesis. Angiography confirmed that all PTFE grafts were patent after 24 h. Doppler flowmetry, performed after 15 and 30 days, showed a reducing patency rate respectively at 70% (21 grafts) and 60% (18 grafts). Morphological studies showed that endothelial lining was not present at 15 day. After 30 days proliferation of blood cells occurred in the graft wall. Lumina presented a fibrous lining and did not show significant endothelial cell growths. These results confirm that PTFE prosthesis represents a suitable alternative to biological graft for the repair of small caliber artery when the latter are not available. Autologous vein is the vascular substitute of choice for peripheral vascular reconstruction, and PTFE prosthesis may be quite successfully used as a secondary choice, when multiple reconstruction are needed and biological grafts are not sufficient.

[Factors influencing the condition of the intima-media complex of carotid and femoral arteries at initial stages of chronic renal failure].

AIM: To examine changes in the structure of large (carotid and femoral) arteries at an early stage of chronic renal failure (CRF) and factors significant for their development. MATERIAL AND METHODS: Duplex ultrasonography of the common carotid arteries (CCA) and common femoral arteries (CFA), serum biochemical tests, echocardiography were made in 32 patients (15 males and 17 females) with chronic diffuse renal disease at an initial stage of CRF (creatinine 2.7 mg%, CRF duration 2.7 years). Increased thickness of the intima-media complex (IMC) in both vascular territories was found in 72% of the examinees. There was a close correlation between CCA and CFA IMC (chi-square = 14.05; p = 0.0002). Plaques in the carotid arteries correlated with smoking (chi-square = 4.60; p = 0.0320), in the femoral arteries--with male sex (chi-square = 5.18; p = 0.0228). IMC of both arteries correlated with age (r = 0.49 and r = 50, respectively, p < 0.05), body mass index (r = 0.50, p < 0.05), thickness of the left ventricular posterior wall and interventricular septum (r = 0.65 and r = 0.55, respectively, p < 0.05), CFA IMC correlated also with creatinine level (r = 0.39, p < 0.05), hypertriglyceridemia (chi-square = 10.33; p = 0.0013), systolic, pulse and mean arterial pressure (r = 0.45, r = 0.38, r = 0.36, respectively, p < 0.05), smoking (r = 0.48, r = 0.40, respectively, p < 0.05) and family history of cardiovascular diseases (chi-square = 7.16; p = 0.0075). A linear multifactorial regression analysis has detected that an independent factor of increased CCA and CFA IMC in patients under 50 years of age was creatinine, in patients over 50 years--age. CONCLUSION: Even at early stages of renal failure patients have thicker IMC associated with both standard risk factors (age, hypertension, smoking, lipid disbolism) and development of renal failure itself.

Intraluminal application of vascular endothelial growth factor enhances healing of microvascular anastomosis in a rat model.

BACKGROUND: Early reconstitution after injury to the endothelium is an important feature for reducing a number of vessel wall pathologies. We investigated the effect of vascular endothelial growth factor (VEGF) and its impact on the vascular remodeling process and reendothelialization after microsurgery. METHODS AND RESULTS: Microvascular anastomosis was performed in the rat femoral artery. One group was treated with intraluminal administration of VEGF and the other with vehicle. We investigated morphological, ultrastructural and immunohistochemical changes of the vascular wall and the reendothelialization process. After 10 days, reendothelialization was significantly faster in VEGF-treated rats. Transmission electron microscopy revealed a complete healing in contrast to vehicle-treated vessels. Moreover, extracellular matrix proteins, such as fibronectin, collagen types I, III and IV, were significantly increased. Furthermore, VEGF treatment significantly induced VEGF receptor 2, flk-1, osteopontin and TGF-beta(1) proteins. CONCLUSIONS: Our data clearly document for the first time that intraluminal treatment with VEGF is beneficial to the healing process in vascular microsurgery. Osteopontin and TGF-beta(1), both induced by VEGF, may play an important role in the vascular remodeling process. Our results provide clear evidence that VEGF application may represent a useful strategy in accelerating reendothelialization and improving vascular healing after microsurgery.

Temporal and spatial characterization of cellular constituents during neointimal hyperplasia after vascular injury: Potential contribution of bone-marrow-derived progenitors to arterial remodeling.

BACKGROUND: Exuberant smooth muscle cells (SMCs) hyperplasia is the major cause of postangioplasty restenosis. We suggested that circulating smooth muscle progenitor cells might contribute to lesion formation after vascular injury. METHODS: We extensively investigated the cellular constituents during neointimal formation after mechanical vascular injury. RESULTS: A large wire was inserted into the mouse femoral artery, causing complete endothelial denudation and marked enlargement of the lumen with massive apoptosis of medial SMCs. At 2 h, the injured artery remained dilated with a thin media containing very few cells. A scanning electron microscopy showed fibrin and platelet deposition at the luminal side. One week after the injury, CD45-positive hematopoietic cells accumulated at the luminal side. Those CD45-positive cells gradually disappeared, whereas neointimal hyperplasia was formed with alpha-smooth muscle actin (SMA) positive cells. Bone marrow cells and peripheral mononuclear cells differentiated into alpha-SMA-positive cells in the presence of PDGF and basic FGF. Moreover, in bone marrow chimeric mice, bone-marrow-derived cells substantially contributed to neointimal hyperplasia after wire injury. CONCLUSION: These results suggest that early accumulation of hematopoietic cells may play a role in the pathogenesis of SMC hyperplasia under certain circumstances.

Increased vascular wall thrombogenicity combined with reduced blood flow promotes occlusive thrombus formation in rabbit femoral artery.

OBJECTIVE: Plaque disruption does not always result in complete thrombotic occlusion. The mechanism of arterial thrombus propagation remains unclear. METHODS AND RESULTS: We studied how vascular wall thrombogenicity and blood flow reduction affect thrombus propagation using a rabbit model of single and repeated balloon injury. After balloon injury of the normal femoral artery, the blood flow was reduced to 50%, 25%, or 10% (n=5). Small mural thrombi composed of aggregated platelets were produced, but no occlusive thrombi developed in any flow reduction. Three weeks after the first balloon injury, neointima with tissue factor expression and increased procoagulant activity was developed. Balloon injury of the neointima with the same blood flow reduction (n=5) induced fibrin-rich thrombus formation. Additionally, injury with flow reduced to 25% and 10% promoted thrombus propagation resulting in vessel occlusion within 160+/-18 and 71+/-17 seconds, respectively. An injection of anti-von Willebrand factor (vWF) monoclonal antibody (AJW200; 1.0 mg/kg) prevented occlusive thrombus formation. CONCLUSIONS: Increased vascular wall thrombogenicity together with a substantial blood flow reduction is crucial for occlusive thrombus formation, and vWF plays an important role in thrombus propagation. Reduced blood flow at plaque disruption sites might contribute to thrombus propagation leading to acute coronary syndromes.

Side-to-side sutureless vascular anastomosis with magnets.

OBJECTIVE: Abbe and Payr introduced vascular techniques and devices to facilitate vessel anastomosis over a century ago. Obora published the idea of a sutureless vascular anastomosis with use of magnetic rings in 1978. The purpose of this study was to assess the performance of a new magnetic device to perform a side-to-side arteriovenous anastomosis in a dog model. MATERIAL AND METHODS: Male fox hounds (25 kg) were treated preoperatively and daily postoperatively with clopidogrel bisulfate (Plavix) and aspirin. The femoral artery and vein were exposed unilaterally in 3 dogs and bilaterally in 4 dogs (n = 11 anastomoses). A 4-mm arteriotomy was performed, and 1 oval magnet 0.5 mm thick was inserted into the lumen of the artery and a second magnet was applied external to the artery, compressing and stabilizing the arterial wall to create a magnetic port. An identical venous magnetic port was created with another pair of oval magnets. When the 2 ports were allowed to approach each other, they self-aligned and magnetically coupled to complete the arteriovenous anastomosis. Patency was assessed for the first hour with direct observation, again after 9 weeks with duplex ultrasound scanning, and at 10 weeks under direct open observation. The anastomoses were explanted after 10 weeks. Hydrodynamic resistance was measured ex vivo on the final 8 anastomoses by measuring the pressure drop across an anastomosis with a known flow rate. RESULTS: After implantation, very high flow created visible turbulence and palpable vibration. All 11 anastomoses were patent under direct observation and palpation. Ten of 11 anastomoses were clearly patent on duplex scans, and patency of 1 anastomosis was questionable. Hydrodynamic resistance averaged 0.73 +/- 0.33 mm Hg min/mL (mean +/- SEM). CONCLUSIONS: Vascular anastomoses performed with magnets demonstrated feasibility; exhibited 100% patency after 10 weeks in a dog arteriovenous shunt model; lacked apparent aneurysm or other potentially catastrophic failure; demonstrated remodeling of the vessel wall after several weeks to incorporate the magnets, making the magnetic force unnecessary; and warrants further study in vessels with different sizes, flow rates, and locations. CLINICAL RELEVANCE: We present a magnet-based device used to perform side-to-side peripheral vascular anastomoses. Its advantages include the ability to anastomose vessels without requiring circumferential surgical exposure. Vascular anastomosis performed with these magnets demonstrated 100% patency in the dog, lacked apparent aneurysm or other potentially catastrophic failure, and demonstrated remodeling of the vessel wall after several weeks, to incorporate the magnets, making indefinite retention of field strength unnecessary. This technique could enable minimally invasive procedures, such as complex reconstructive and revascularizing surgery, and warrants further study in vessels with different sizes, flow rates, and locations.

Collateral artery growth (arteriogenesis) after experimental arterial occlusion is impaired in mice lacking CC-chemokine receptor-2.

Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. Induced macrophage migration in vivo is driven by the binding of monocyte chemoattractant protein-1 (MCP-1, or CCL2 in the new nomenclature) to the CCR2-chemokine receptor on macrophages. To determine whether the CCL2-CCR2 signaling pathway is involved in the accumulation of macrophages in growing collateral vessels, we used mice that are deficient in CCR2 in a model of experimental arterial occlusion and collateral vessel growth. In an in vitro CCL2-driven chemotaxis assay, mononuclear cells isolated from wild-type BALB/c mice exhibited CCL2 concentration-dependent migration, whereas this migration was abolished in cells from CCR2(-/-) mice on a BALB/c genetic background. In vivo, blood flow recovery as measured by laser Doppler (LDI) and MRI (MRI) was impaired in CCR2(-/-) mice on either the BALB/c or C57BL/6 genetic backgrounds. Three weeks after femoral artery ligation, LDI perfusion ratio of operated versus nonoperated distal hindlimb in BALB/c wild-type mice increased to 0.45+/-0.06 and in CCR2(-/-) animals only to 0.21+/-0.03 (P<0.01). In C57BL/6 mice, ratio increased to 0.96+/-0.09 and 0.85+/-0.08 (P<0.05), respectively. MRI at 3 weeks (0.76+/-0.06 versus 0.62+/-0.01; P<0.05) and hemoglobin oxygen saturation measurements confirmed these findings. Active foot movement score significantly decreased and gastrocnemius muscle atrophy was significantly greater in CCR2(-/-) mice. Morphometric analysis showed a lesser increase in collateral vessel diameters in CCR2(-/-) mice. Importantly, the number of invaded monocytes/macrophages in the perivascular space of collateral arteries of CCR2(-/-) animals was dramatically reduced in comparison to wild-type mice. In conclusion, our results demonstrate that the CCR2 signaling pathway is essential for efficient collateral artery growth.

C5b-9 terminal complement complex assembly on apoptotic cells in human arterial wall with atherosclerosis.

Apoptosis plays an important role in atherosclerosis. The factors regulating this process are not well defined. We examined the relation of apoptotic cells with the terminal complement complex C5b-9 in human atherosclerotic lesions. The extent of apoptosis was determined using TdT dUTP nick-end labeling (TUNEL) and immunohistochemistry of apoptosis regulators caspase-3, caspase-9, Bax, and Bcl-2. C5b-9 was localized by immunohistochemistry and immunoelectron microscopy. The apoptotic index was higher in fibrous plaques when compared with intimal fatty streaks and intimal thickenings. Bax expression was present in TUNEL+ apoptotic cells, and Bcl-2 was rarely present in the atherosclerotic wall. Active caspase 9 and caspase 3 deposits were present in the same areas, suggesting an involvement of the mitochondrial pathway. C5b-9 deposits colocalized with TUNEL+ cells, and the percent of double-positive cells was 2% in fatty streaks, 12% in intimal thickenings, and 35% in fibrous plaques. Colocalization of apoptotic cells with C5b-9 was also confirmed by immunoelectron microscopy. In conclusion, some apoptotic cells carry C5b-9 deposits, suggesting that complement might be activated by apoptotic cells and involved in the promotion of apoptosis, contributing to the progression of atherosclerotic lesions.

Challenging Alexis Carrel: a study in alternate microvascular techniques.

INTRODUCTION: Alexis Carrel pioneered the full-thickness triangulated vascular repair technique, which led to a Nobel Prize in 1912. However, microvascular anastomotic techniques that do not violate the intima, such as the VCS microclip repair and partial-thickness suturing, limit trauma to the intima, thus minimizing the potential for thrombosis. Our study compares such techniques with the standard full thickness-anastomotic repair. METHODS: Thirty-two end-to-end anastomotic repairs were performed in rat femoral arteries 1 mm or less in diameter. Group I: thirteen full-thickness repairs were completed using 10-0 nylon on a BV75 microm needle. Nineteen extraluminal repairs were performed using either a partial thickness technique with an 11-0 nylon BV50 microm needle (Group II, n = 12) or VCS nonpenetrating clip (Group III, n=7). Casted samples, injected with methylmethacrylate, were harvested at 1 and 3 weeks for histopathological evaluation. The presence of thrombosis, inflammation, endothelialization, angiogenesis and intimal hyperplasia were described for each repair. RESULTS: Statistical analysis revealed no difference between the intraluminal and extraluminal techniques. Patency rates were similar between both groups: 92% (12/13) for Group I and 94% (17/18) for the extraluminal Groups II and III combined. One-hundred per cent of partial thickness suture repairs were patent. Histology revealed localized inflammation to the adventitia and media, as well as endothelialization at 1 week for anastomoses in Groups II and III. The intima of Group I demonstrated proliferative characteristics in contrast to the extraluminal groups, where secretory myofibroblasts were prevalent. The anastomotic microcirculation did not originate from the repaired artery in any of the groups. CONCLUSION: Patency rates with end-to-end anastomotic repairs using a partial thickness technique are comparable to the standard full-thickness technique. Repairs that do not include the intima revealed focal inflammatory responses to the outer layers and more rapid endothelialization, while neighboring vessels perfuse the healing anastomosis.

Expression of ICAM-1, VCAM-1, E-selectin and TNF-alpha on the endothelium of femoral and iliac arteries in thromboangiitis obliterans.

Immunohistochemical light and electron microscopical analysis of surgical biopsies obtained from femoral and iliac arteries of patients with thromboangiitis obliterans (TAO) were performed to investigate the presence of tumour necrosis factor-alpha (TNF-alpha) and expression of the endothelial cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Expression of ICAM-1, VCAM-1 and E-selectin was increased on endothelium and some inflammatory cells in the thickened intima in all TAO patients. Ultrastructural immunohistochemistry revealed contacts between mononuclear blood cells and ICAM-1-, and E-selectin-positive endothelial cells. These endothelial cells showed morphological signs of activation. The present data indicate that endothelial cells are activated in TAO and that vascular lesions are associated with TNF-alpha secretion by tissue-infiltrating inflammatory cells, ICAM-1-, VCAM-1- and E-selectin expression on endothelial cells and leukocyte adhesion via their ligands. The preferential expression of inducible adhesion molecules in microvessels and mononuclear inflammatory cells suggests that angiogenesis contributes to the persistence of the inflammatory process in TAO.

Color-flow duplex scanning of the leg arteries by use of a new echo-enhancing agent.

OBJECTIVE: This was a dose-finding and effectiveness study of a newly developed contrast-enhancing agent, sulphur hexafluoride (SF(6)), in patients with peripheral arterial disease in whom the therapeutic policy could not be established on the basis of standard color-flow duplex scanning of the leg arteries. METHODS: In this open-label, randomized, dose-ranging, crossover design, 14 patients in whom the assessment of vessel patency was difficult because of poor visibility (low-flow state) or extensive wall calcifications were studied. Contrast-enhanced duplex scanning was performed on the upper leg (n = 4), lower leg (n = 6), or pedal (n = 4) arteries after intravenous injection of four different dosages of SF(6). The results were compared with those from selective angiography of the vessel of interest. Contrast duration and agreement about the diagnosis and the confidence in the diagnosis were obtained before and after administration of the contrast agent. RESULTS: No adverse effects of the contrast agent were seen. Overall agreement was reasonable with regard to vessel patency between contrast-enhanced duplex scanning and angiography (71%). Nine of 14 vessels (64%) appeared open when contrast was applied. In four cases this could not be confirmed by angiography; in two of these cases this was due to the presence of collateral vessels. All vessels that appeared occluded with the contrast agent were also occluded on the angiogram. The confidence in the diagnosis increased from 56% to 91% after contrast administration (P <.0001). CONCLUSION: SF(6)-enhanced color-flow duplex scanning is a safe method that may improve the assessment of the patency of leg arteries, particularly in low-flow states. The visualization of collateral vessles during (enhanced) duplex scanning may be misleading because they may be regarded as the vessel of interest.

Topically administered macromolecular heparin proteoglycans inhibit thrombus growth in microvascular anastomoses.

Previously, during blood perfusion over collagen-coated surfaces; soluble or immobilized heparin proteoglycans (HEP-PG) have been shown to block thrombus growth. Our aim was to study the antithrombotic effect of locally applied unfractionated heparin (UFH, 1 mg/ml), or rat mast cell-derived HEP-PG (MW 750 kD, 10 microg/ml) compared with saline in early (10 min) and late (3 days) thrombus formation upon anastomosis of rat common femoral arteries. In both semiquantitative scanning electron microscopy (SEM) and quantitative platelet Indium 111-labeling HEP-PG inhibited thrombus growth in comparison with saline. At 10 min, the extent of thrombosis (scale 1-4) in SEM followed the order: saline (3.2+/-0.8) > UFH (2.8+/-1.0) > HEP-PG (1.8+/-0.8), and also Indium 111-positive platelets (10(6)) accumulated on the anastomosed vessel in the same order 14.2 +/-7.2, 10.3 +/-5.0, and 7.7 +/-3.1 (saline vs. HEP-PG, p = 0.03 and 0.05, respectively). At 3 days all HEP-PG-treated vessels remained patent with only small mural thrombi, whereas 2/7 saline- and 1/7 UFH-treated anastomoses occluded and showed more thrombosis overall. We conclude that locally administered HEP-PG inhibit arterial thrombus growth in anastomosed small-sized arteries and could prevent thrombotic complications in (micro)vascular surgery and arteriovenous shunts.

[Scanning electronic microscope observation of elastic fiber of anastomosed artery].

OBJECTIVE: To observe the architecture of elastic fiber of anastomosed artery. METHODS: The right femoral arteries of 60 Wistar rats were cut off transversely and end-to-end anastomosis were performed. On the 3rd, 7th, 14th, 21st, 30th and 90th days after operation, the anastomosed artery segments were harvested and fixed by 10% formalin. After routine processed, the architecture of elastic fiber of anastomosed artery was observed under scanning electronic microscope and was compared with that of normal artery. RESULTS: On the 3rd and 7th days after anastomosis, there was no the elastic fiber in the middle of the anastomosed area. From 14 to 90 days after anastomosis, the newborn elastic fiber connected the anastomosed area. The reconstruction of elastic fiber could be divided into quiescent stage, proliferation stage, and rebuilding stage. CONCLUSION: The reconstruction of elastic fiber occurs after arterious anastomosis and newborn elastic fiber originates from endoarterious layer. The structure of elastic fiber can return to normal 30 days after anastomosis.