Effects of graft preservation conditions on coronary endothelium and cardiac functional recovery in a rat model of donation after circulatory death.

BACKGROUND: Use of cardiac grafts obtained with donation after circulatory death (DCD) could significantly improve donor heart availability. As DCD hearts undergo potentially deleterious warm ischemia and reperfusion, clinical protocols require optimization to ensure graft quality. Thus, we investigated effects of alternative preservation conditions on endothelial and/or vascular and contractile function in comparison with the current clinical standard. METHODS: Using a rat DCD model, we compared currently used graft preservation conditions, St. Thomas n°2 (St. T) at 4°C, with potentially more suitable conditions for DCD hearts, adenosine-lidocaine preservation solution (A-L) at 4°C or 22°C. Following general anesthesia and diaphragm transection, hearts underwent either 0 or 18 min of in-situ warm ischemia, were explanted, flushed and stored for 15 min with either St. T at 4°C or A-L at 4°C or 22°C, and then reperfused under normothermic, aerobic conditions. Endothelial integrity and contractile function were determined. RESULTS: Compared to 4°C preservation, 22°C A-L significantly increased endothelial nitric oxide synthase (eNOS) dimerization and reduced oxidative tissue damage (p < 0.05 for all). Furthermore, A-L at 22°C better preserved the endothelial glycocalyx and coronary flow compared with St. T, tended to reduce tissue calcium overload, and stimulated pro-survival signaling. No significant differences were observed in cardiac function among ischemic groups. CONCLUSIONS: Twenty-two-degree Celsius A-L solution better preserves the coronary endothelium compared to 4°C St. T, which likely results from greater eNOS dimerization, reduced oxidative stress, and activation of the reperfusion injury salvage kinase (RISK) pathway. Improving heart preservation conditions immediately following warm ischemia constitutes a promising approach for the optimization of clinical protocols in DCD heart transplantation.

Ultrastructural Investigations of Arterial Bypass Conduits after the Use of Different Harvesting Techniques Using an Electron Microscope.

OBJECTIVES: The aim of this study was to perform morphometric analysis of arterial conduits harvested by harmonic scalpel in coronary artery bypass grafting (CABG) patients. METHODS: From 100 CABG patients, 200 arterial conduits-100 radial arteries (RAs) and 100 left internal thoracic artery (LITAs)-were harvested. The patients had similar characteristics (mean age, sex ratio, comorbidities, etc.). We divided the patients into 2 groups according to harvesting technique. In group 1, a harmonic scalpel was used in 50 patients for harvesting arterial conduits (50 LITA and 50 RA). In group 2, conduits were harvested using low-voltage electrocautery. To prevent side effects of clipping, all conduits in both groups remained in perfused condition until anastomosis. A 10-mm length of conduit was cut for transmission electron microscopy investigation. We calculated duration of harvesting, blood flow changes, and histopathologic changes of the conduits according to a vessel scoring system. RESULTS: In the harmonic scalpel group, we detected pathologic findings-corruption of endothelial integrity, subendothelial damage, and endothelial pathology-in 5 specimens (10%) (3 LITA  [6%] and 2 RA [4%]). In group 2, pathologic findings were detected in 16 conduits (32%; 11 LITA, 22%, and 5 RA, 10%). Endothelial dissection, subendothelial disarrangement, cellular separation due to intercellular edema, and subadventitial hematoma were the main pathologic changes in the classic harvesting method. There was a significant difference between the groups (P = .001). Harvesting time of LITA was nearly similar in both groups: 26.9 ± 11.1 min (range 25-38) in group 1 and 21.3 ± 8.6 min (range 21-25) in group 2 (P = .049). RA harvesting time was significantly shorter with the harmonic scalpel technique (20.3 ± 3.9 versus 27.6 ± 5.4 min, P = .022). The blood flow of the conduits was similar, with no statistical difference for the 2 arterial conduits (LITA, P = .76; RA, P = .55). CONCLUSION: In the learning curve period, the use of a harmonic scalpel is time consuming and presents some difficulties during the harvesting of conduits. According to our study results, however, the harmonic scalpel technique may be useful because of decreased pathology, including spasm. In our opinion, graft occlusion or thrombus as a life-threatening condition and endothelial dysfunction may decrease with the use of this alternative harvesting technique.

Surgical Preparation Reduces Hydrogen Sulfide Released from Human Saphenous Veins in Coronary Artery Bypass Grafting.

The long-term patency rate of saphenous vein (SV) grafts is poor compared to arterial grafts. To investigate the effects of surgical preparation (distention) of SV on hydrogen sulfide (H2S) released from the endothelium, human SV segments were harvested from 43 patients during coronary artery bypass surgery (CABG). Acetylcholine (ACh) induced relaxation that was inhibited by NG-nitro-L-arginine + indomethacin and cysteine aminotransferase inhibitor aminooxyacetic acid in the normal SV. In contrast, ACh did not evoke relaxation in the distended SV (DSV). The concentration of H2S quantified by methylene blue assay in DSV was significantly lower than that in control. Transmission electron microscope and immunohistochemistry studies showed that the preparation destroyed the endothelium, smooth muscle, organelle, and vasa vasorum. We conclude that surgical preparation injures the endothelium and smooth muscle of the SV grafts and reduces H2S release from SV. These effects may contribute to the poor long-term patency of the SV graft.

Factors influencing corneal predescemetic endothelial keratoplasty (PDEK) graft creation: It's all in a bubble.

AIM: To assess the effect of pneumatic (air) and fluidic (transport medium) injection to the type of bubble (I, II or mixed III) and the resultant dissection of corneal endothelial grafts PDEK or DMEK. MATERIALS AND METHODS: All grafts were obtained from Dr Agrawal Hospital's Eye Bank. Air injection was the initial preferred mode of graft harvest. If pneumatic dissection was unsuccessful after 10 tries, fluidic dissection with transport medium was tried. SPSS 23.0 was used to statistically analyse the data. RESULTS: 40 consecutive donor corneas with a mean age of 46.5 and a mean endothelial count of 2980 were analysed. Air dissection lead to the harvest of 27 endothelial grafts and fluid dissection led to the creation of 7 endothelial grafts. Statistically significant difference was found the different bubble types and the type of injection (χ2 chi square=10.02, 0=0.008). CONCLUSION: In young donors pneumatic (air) graft dissection leads to PDEK in a high proportion. This percentage is reduced when transport medium is tried after unsuccessful air injection. Injection of transport medium increases the percentage of grafts harvested but also increases the ratio of Type II and III DMEK grafts created.

Allogenic endothelial progenitor cell transplantation increases flap survival through an upregulation of eNOs and VEGF on venous flap survival in rabbits.

BACKGROUND: Endothelial progenitor cells (EPCs) are one type of bone marrow hematopoietic stromal cells which play a vital role in neovascularization and tissue repair. In this study, we investigated whether EPCs promote flap survival in a rabbit venous model. MATERIALS AND METHODS: EPCs were customized from CHI Scientific, Inc, China. Thirty-six rabbits were randomly assigned to either the sham group (n = 12), the control group (n = 12) or the EPC transplantation group (n = 12). A 10 × 6 cm venous flap was created on the rabbit abdomen. Both the EPC transplantation and control groups had the same volume of EPCs-PBS (phosphate buffered saline) and PBS on postoperative day 1. Flap survival, blood flow, histopathology, expression of endothelial nitric oxide synthase (eNOs) and Vascular Endothelial Growth Factor (VEGF) were detected on postoperative day 10. RESULTS: Cellular immunofluorescence assay positively confirmed that the EPCs were undergoing differentiation. The survival rate of the flap in the EPC transplantation group was 58.4 ± 7.1%, which was significantly higher than that of the control group (4.8 ± 3.4%) (p<0.01). Histological examination revealed that the EPC transplantation group had higher microvessel density, fewer inflammatory cells, and a higher expression of eNOs and VEGF. Significantly increased blood flow perfusion was seen in the EPC transplantation group using laser Doppler imaging. The Western Blot technique revealed that the expression of eNOs and VEGF in the EPC transplantation group were both significantly higher than those in the control group. CONCLUSION: This study demonstrated that EPC transplantation improved venous flap survival in rabbits. The present findings may provide insight into the promotion of venous flap survival in clinical practice in the future.

Enhanced protection of the renal vascular endothelium improves early outcome in kidney transplantation: Preclinical investigations in pig and mouse.

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.

Randomized Study Comparing the Effect of Carbon Dioxide Insufflation on Veins Using 2 Types of Endoscopic and Open Vein Harvesting.

OBJECTIVE: The aim of the study was to assess whether the use of carbon dioxide insufflation has any impact on integrity of long saphenous vein comparing 2 types of endoscopic vein harvesting and traditional open vein harvesting. METHODS: A total of 301 patients were prospectively randomized into 3 groups. Group 1 control arm of open vein harvesting (n = 101), group 2 closed tunnel (carbon dioxide) endoscopic vein harvesting (n = 100) and Group 3 open tunnel (carbon dioxide) endoscopic vein harvesting (open tunnel endoscopic vein harvesting) (n = 100). Each group was assessed to determine the systemic level of partial arterial carbon dioxide, end-tidal carbon dioxide, and pH. Three blood samples were obtained at baseline, 10 minutes after start of endoscopic vein harvesting, and 10 minutes after the vein was retrieved. Vein samples were taken immediately after vein harvesting without further surgical handling to measure the histological level of endothelial damage. A modified validated endothelial scoring system was used to compare the extent of endothelial stretching and detachment. RESULTS: The level of end-tidal carbon dioxide was maintained in the open tunnel endoscopic vein harvesting and open vein harvesting groups but increased significantly in the closed tunnel endoscopic vein harvesting group (P = 0.451, P = 0.385, and P < 0.001). Interestingly, partial arterial carbon dioxide also did not differ over time in the open tunnel endoscopic vein harvesting group (P = 0.241), whereas partial arterial carbon dioxide reduced significantly over time in the open vein harvesting group (P = 0.001). A profound increase in partial arterial carbon dioxide was observed in the closed tunnel endoscopic vein harvesting group (P < 0.001). Consistent with these patterns, only the closed tunnel endoscopic vein harvesting group demonstrated a sudden drop in pH over time (P < 0.001), whereas pH remained stable for both open tunnel endoscopic vein harvesting and open vein harvesting groups (P = 0.105 and P = 0.869, respectively). Endothelial integrity was better preserved in the open vein harvesting group compared with open tunnel endoscopic vein harvesting or closed tunnel endoscopic vein harvesting groups (P = 0.012) and was not affected by changes in carbon dioxide or low pH. Significantly greater stretching of the endothelium was observed in the open tunnel endoscopic open tunnel endoscopic vein harvesting group compared with the other groups (P = 0.003). CONCLUSIONS: This study demonstrated that the different vein harvesting techniques impact on endothelial integrity; however, this does not seem to be related to the increase in systemic absorption of carbon dioxide or to the pressurized endoscopic tunnel. The open tunnel endoscopic harvesting technique vein had more endothelial stretching compared with the closed tunnel endoscopic technique; this may be due to manual dissection of the vein. Further research is required to evaluate the long-term clinical outcome of these vein grafts.

Endothelial Function Is Preserved in Veins Harvested by Either Endoscopic or Surgical Techniques.

BACKGROUND: Endoscopic vein harvest for lower extremity arterial bypass grafting has been questioned due to concern for endothelial damage during procurement. We sought to compare nitric oxide (NO)-mediated endothelial-dependent relaxation (EDR) in vein segments harvested using open surgical techniques (OH) versus endoscopic vein harvest (EH) techniques. METHODS: Saphenous vein segments were harvested for lower extremity bypass, and a single, minimally handled section of saphenous vein, free of branches, was taken from the end of the graft. Four 4-mm venous ring segments were then cut and mounted on force transducers. Segments were mounted in 37° oxygenated Krebs-Henseleit solution and maximally contracted using KCl. Individual ring segments that did not react to KCl were excluded from the study. Norepinephrine (NE) was used to achieve submaximal contraction. EDR was determined using increasing concentrations of bradykinin (BDK). Endothelial-independent relaxation (EIR) was confirmed using sodium nitroprusside. Two-way analysis of variance (ANOVA) was used to analyze differences between harvest techniques across BDK concentration and a Student's t-test was used to analyze single comparisons. RESULTS: Vein segments harvested from patients (n = 13) led to 28 viable rings that exhibited a positive reaction to KCl (11 rings; 5 patients EH vs. 17 rings; 8 patients OH). Both vein groups achieved moderate relaxation to maximal BDK concentration, [10-6 M]; (49.5% EH vs. 40.55% OH, P = 0.270). Analysis by 2-way ANOVA for mean % relaxation for BDK concentration [10-11-10-6 M] showed improved EDR in EH samples compared with OH (P = 0.029). Mean nitrite/nitrate (NO(x)) tissue bath concentration measurements post-BDK were 139.8 nM (EH) vs. 97.2 nM (OH; P = 0.264). Histology and positive factor VIII immunohistochemistry staining provided evidence for the presence of intact endothelium in our sample segments. EIR was preserved and was similar in the two groups. CONCLUSIONS: Endothelial function is preserved when utilizing endoscopic harvesting techniques. The advantages of minimally invasive vein procurement for lower extremity bypass can be obtained without concern for damaging venous endothelium.

Endothelial colony-forming cells ameliorate endothelial dysfunction via secreted factors following ischemia-reperfusion injury.

Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFC) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFC may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia-reperfusion (I/R). Rat pulmonary microvascular endothelial cells (PMVEC) with high proliferative potential were compared with pulmonary artery endothelial cells (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 h of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of ICAM-1 and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function.

Identification of blood vascular endothelial stem cells by the expression of protein C receptor.

Vascular growth and remodeling are dependent on the generation of new endothelial cells from stem cells and the involvement of perivascular cells to maintain vessel integrity and function. The existence and cellular identity of vascular endothelial stem cells (VESCs) remain unclear. The perivascular pericytes in adult tissues are thought to arise from the recruitment and differentiation of mesenchymal progenitors during early development. In this study, we identified Protein C receptor-expressing (Procr+) endothelial cells as VESCs in multiple tissues. Procr+ VESCs exhibit robust clonogenicity in culture, high vessel reconstitution efficiency in transplantation, long-term clonal expansion in lineage tracing, and EndMT characteristics. Moreover, Procr+ VESCs are bipotent, giving rise to de novo formation of endothelial cells and pericytes. This represents a novel origin of pericytes in adult angiogenesis, reshaping our understanding of blood vessel development and homeostatic process. Our study may also provide a more precise therapeutic target to inhibit pathological angiogenesis and tumor growth.

Neovascularization Potential of Blood Outgrowth Endothelial Cells From Patients With Stable Ischemic Heart Failure Is Preserved.

BACKGROUND: Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models, but outgrowth characteristics and functionality of BOECs from patients with ischemic cardiomyopathy (ICMP) are unknown. We compared outgrowth efficiency and in vitro and in vivo functionality of BOECs derived from ICMP with BOECs from age-matched (ACON) and healthy young (CON) controls. METHODS AND RESULTS: We isolated 3.6±0.6 BOEC colonies/100×10(6) mononuclear cells (MNCs) from 60-mL blood samples of ICMP patients (n=45; age: 66±1 years; LVEF: 31±2%) versus 3.5±0.9 colonies/100×10(6) MNCs in ACON (n=32; age: 60±1 years) and 2.6±0.4 colonies/100×10(6) MNCs in CON (n=55; age: 34±1 years), P=0.29. Endothelial lineage (VEGFR2(+)/CD31(+)/CD146(+)) and progenitor (CD34(+)/CD133(-)) marker expression was comparable in ICMP and CON. Growth kinetics were similar between groups (P=0.38) and not affected by left ventricular systolic dysfunction, maladaptive remodeling, or presence of cardiovascular risk factors in ICMP patients. In vitro neovascularization potential, assessed by network remodeling on Matrigel and three-dimensional spheroid sprouting, did not differ in ICMP from (A)CON. Secretome analysis showed a marked proangiogenic profile, with highest release of angiopoietin-2 (1.4±0.3×10(5) pg/10(6) ICMP-BOECs) and placental growth factor (5.8±1.5×10(3) pg/10(6) ICMP BOECs), independent of age or ischemic disease. Senescence-associated ß-galactosidase staining showed comparable senescence in BOECs from ICMP (5.8±2.1%; n=17), ACON (3.9±1.1%; n=7), and CON (9.0±2.8%; n=13), P=0.19. High-resolution microcomputed tomography analysis in the ischemic hindlimb of nude mice confirmed increased arteriogenesis in the thigh region after intramuscular injections of BOECs from ICMP (P=0.025; n=8) and CON (P=0.048; n=5) over vehicle control (n=8), both to a similar extent (P=0.831). CONCLUSIONS: BOECs can be successfully culture-expanded from patients with ICMP. In contrast to impaired functionality of ICMP-derived bone marrow MNCs, BOECs retain a robust proangiogenic profile, both in vitro and in vivo, with therapeutic potential for targeting ischemic disease.

Preservation of Endothelial and Smooth Muscle Function of Human Saphenous Vein Transplants.

OBJECTIVES: Methods for conservation and preservation of vascular grafts are often controversially discussed. Furthermore, immunologic monitoring or immunotherapy for allogeneic graft is not considered necessary in many cases. The present study was initiated to examine the cellular vitality and functional efficiency of vein transplant during preservation. MATERIALS AND METHODS: Twenty-seven human vein segments (vena saphena magna) were stored after explant in University of Wisconsin solution or histidine-tryptophan-ketoglutarate solution at 4 °C. After 3, 24, 48, 72, and 96 hours, vein functionality was tested. Ring segments were fixed by triangles in Krebs-Henseleit buffer. Contractile function was measured after addition of potassium chloride solution (80 mM) and phenylephrine (0.2, 2, or 20 µM). To investigate endothelium-dependent vasorelaxation, 1 µM acetylcholine was added. RESULTS: Of 27 segments, 5 showed endothelium-dependent relaxation. Vasorelaxation continued for up to 48 hours after administration of acetylcholine in University of Wisconsin solution and for up to 24 hours in histidine-tryptophane-ketoglutarate solution. At 48 hours, potassium chloride solution-induced vasocontraction was 17% more effective than phenylephrine in University of Wisconsin solution. University of Wisconsin solution was significantly more effective than histidine-tryptophane-ketoglutarate solution in terms of preservation of phenylephrine (0.2, 2 µM)-induced vasocontraction. Phenylephrine (2 µM)-induced contraction was retained in University of Wisconsin solution after 24 hours by 81% and after 48 hours by 55%, with comparable results in histidine-tryptophane-ketoglutarate solution of only 62% and 34% after 24 and 48 hours. CONCLUSIONS: At 48 hours, human saphenous vein transplants had better endothelium and smooth muscle function when preserved in University of Wisconsin solution versus histidine-tryptophane-ketoglutarate solution.

Coimplanted endothelial cells improve adipose tissue grafts' survival by increasing vascularization.

BACKGROUND: With goal of improving fat graft survival, many studies have focused on supplementing cells in the graft fat. In these studies, enhanced vascularization is considered the most important mechanism for the improved graft survival. Endothelial cells (ECs) are essential in vessel formation of the vascularization. Therefore, in this study, we coimplanted ECs with adipose tissue to investigate whether the ECs can enhance graft survival in a cell concentration-dependent manner. METHODS: Endothelial cells were isolated from stromal vascular fraction derived from human liposuction aspirates, and the EC characteristics were confirmed by CD31 immunofluorescence staining, measuring acetylated low-density lipoprotein uptake, and observing the formation of capillary-like tubular structures in Matrigel. During the animal experiment, the isolated ECs were labeled, then added to 0.5-mL fat grafts at different numbers (0.5 × 10(6), 1 × 10(6), 2 × 10(6), and 4 × 10(6) cells) before subcutaneous implantation in nude mice. Grafts were harvested at 1 week, 1 month, and 2 months after -transplantation, and graft survival and vascularization were evaluated based on weight measurements, histological assessment, and vascular gene expression. RESULTS: Stromal vascular fraction-derived vascular cells exhibited typical EC characteristics. The observed differences in explanted graft weight, vessel density, vascular gene expression, and cell tracking result indicated that coimplantation with ECs accelerated vascularization that increased graft survival in a concentration-dependent manner. Over the experimental period, fat grafts implanted with 4 × 10(6) ECs showed no weight loss and the greatest increases in measures of vascularization. CONCLUSIONS: Endothelial cells can effectively enhance vascularization in fat grafts, and higher EC concentrations (eg, 4 × 10(6) ECs/0.5 mL adipose tissue) may best support graft survival.

Targeted protection of donor graft vasculature using a phosphodiesterase inhibitor increases survival and predictability of autologous fat grafts.

BACKGROUND: Fat grafting is limited by unpredictable long-term graft retention. The authors postulate that injury to the donor-derived microvasculature during harvest and subsequent ischemia may account for this clinical variability. They examined the use of the U.S. Food and Drug Administration-approved phosphodiesterase-5 inhibitor sildenafil citrate to protect graft microvasculature and its role in revascularization and survival. METHODS: Inguinal fat of donor Tie2/LacZ mice was infiltrated with sildenafil or saline, harvested, and transplanted onto the dorsa of recipient FVB mice. Additional donor mice were perfused with intraarterial trypsin to inactivate the fat graft microvasculature before harvest and transplantation. Differences in graft revascularization, perfusion, volume of retention, and biochemical changes were assessed. RESULTS: Surviving fat grafts were characterized by exclusively donor-derived vasculature inosculating with the recipient circulation at the graft periphery. Inactivation of donor-derived microvasculature decreased early graft perfusion and led to nearly total graft loss by 8 weeks. Sildenafil attenuated vascular ischemic injury, consistent with reductions in VCAM-1 and SDF1α expression at 48 hours and 4-fold increases in microvasculature survival by 2 weeks over controls. Compared with controls, targeted sildenafil treatment improved early graft perfusion, doubled graft retention at 12 weeks (83 percent versus 39 percent; p < 0.05), ultimately retaining 64 percent of the original graft volume by 24 weeks (compared to 4 percent; p < 0.05) with superior histologic features. CONCLUSIONS: Fat graft vascularization is critically dependent on maintenance of the donor microvasculature. Sildenafil protects the donor microvasculature during transfer and revascularization, increasing long-term volume retention. These data demonstrate a rapidly translatable method of increasing predictability and durability of fat grafting in clinical practice.

Platelet adhesion on endothelium early after vein grafting mediates leukocyte recruitment and intimal hyperplasia in a murine model.

Intimal hyperplasia (IH) is the substrate for accelerated atherosclerosis and limited patency of vein grafts. However, there is still no specific treatment targeting IH following graft surgery. In this study, we used a mouse model of vein grafting to investigate the potential for early intervention with platelet function for later development of graft IH. We transferred the inferior vena cava (IVC) from donor C57BL/6 mice to the carotid artery in recipients using a cuff technique. We found extensive endothelial injury and platelet adhesion one hour following grafting. Adhesion of leukocytes was distinct in areas of platelet adhesion. Platelet and leukocyte adhesion was strongly reduced in mice receiving a function-blocking antibody against the integrin αIIbß3. This was followed by a reduction of IH one month following grafting. Depletion of platelets using antiserum also reduced IH at later time points. These findings indicate platelets as pivotal to leukocyte recruitment to the wall of vein grafts. In conclusion, the data also highlight early intervention of platelets and inflammation as potential treatment for later formation of IH and accelerated atherosclerosis following bypass surgery.

Blood vessel-derived acellular matrix for vascular graft application.

To overcome the issues connected to the use of autologous vascular grafts and artificial materials for reconstruction of small diameter (<6 mm) blood vessels, this study aimed to develop acellular matrix- (AM-) based vascular grafts. Rat iliac arteries were decellularized by a detergent-enzymatic treatment, whereas endothelial cells (ECs) were obtained through enzymatic digestion of rat skin followed by immunomagnetic separation of CD31-positive cells. Sixteen female Lewis rats (8 weeks old) received only AM or previously in vitro reendothelialized AM as abdominal aorta interposition grafts (about 1 cm). The detergent-enzymatic treatment completely removed the cellular part of vessels and both MHC class I and class II antigens. One month after surgery, the luminal surface of implanted AMs was partially covered by ECs and several platelets adhered in the areas lacking cell coverage. Intimal hyperplasia, already detected after 1 month, increased at 3 months. On the contrary, all grafts composed by AM and ECs were completely covered at 1 month and their structure was similar to that of native vessels at 3 months. Taken together, our findings show that prostheses composed of AM preseeded with ECs could be a promising approach for the replacement of blood vessels.

The performance of a small-calibre graft for vascular reconstructions in a senescent sheep model.

There is an acute clinical need for small-calibre (<6 mm) vascular grafts for surgery. The aim of this study was to evaluate the long-term performance of a small-calibre graft produced from a nanocomposite biomaterial, polyhedral oligomeric silsesquioxane poly(carbonate-urea)urethane (POSS-PCU), in a large animal model following Good Manufacturing Practice (GMP) and Good Laboratory Practice (GLP) protocols. Grafts were characterised and implanted into the left carotid artery (LCA) of senescent sheep (n = 11) for a period of 9 months. In vivo compliance and blood flow rates were measured using ultrasound wall tracking software and a Transonic flow meter. Graft patency and degree of intimal hyperplasia (IH) were examined at the study end point. Seven of the POSS-PCU grafts were free from thrombosis, IH, calcification and aneurysmal dilation, with 4 occluding within 14 days. All of the ePTFE controls (n = 4) were found to be occluded by day 32. The lumen of the patent POSS-PCU grafts was free from any cellular deposits, whilst perigraft tissue could be seen to be infiltrating into the body of the graft from the adventitia. No significant differences were detected between the blood flow rates (p = 0.3693) and compliance (p = 0.9706) of the POSS-PCU grafts and the native artery, either post-operatively or after 9 months implantation. Small-calibre vascular grafts produced from POSS-PCU offer a viable option for the clinical use in revascularisation procedures with a patency rate of 64%.

Loss of mural cells leads to wall degeneration, aneurysm growth, and eventual rupture in a rat aneurysm model.

BACKGROUND AND PURPOSE: The biological mechanisms predisposing intracranial saccular aneurysms to growth and rupture are not yet fully understood. Mural cell loss is a histological hallmark of ruptured cerebral aneurysms. It remains unclear whether mural cell loss predisposes to aneurysm growth and eventual rupture. METHODS: Sodium dodecyl sulfate decellularized and nondecellularized saccular aneurysm from syngeneic thoracic aortas were transplanted to the abdominal aorta of Wistar rats. Aneurysm patency and growth was followed up for 1 month with contrast-enhanced serial magnetic resonance angiographies. Endoscopy and histology of the aneurysms were used to assess the role of periadventitial environment, aneurysm wall, and thrombus remodeling. RESULTS: Nondecellularized aneurysms (n=12) showed a linear course of thrombosis and remained stable. Decellularized aneurysms (n=12) exhibited a heterogeneous pattern of thrombosis, thrombus recanalization, and growth. Three of the growing aneurysms (n=5) ruptured during the observation period. Growing and ruptured aneurysms demonstrated marked adventitial fibrosis and inflammation, complete wall disruption, and increased neutrophil accumulation in unorganized intraluminal thrombus. CONCLUSIONS: In the presented experimental setting, complete loss of mural cells acts as a driving force for aneurysm growth and rupture. The findings suggest that aneurysms missing mural cells are incapable to organize a luminal thrombus, leading to recanalization, increased inflammatory reaction, severe wall degeneration, and eventual rupture.

Addition of vardenafil into storage solution protects the endothelium in a hypoxia-reoxygenation model.

OBJECTIVE: Based upon the well known protective effect of intracellular cyclic guanosine monophosphate (cGMP) accumulation, we tested the hypothesis that storage solution enriched with optimal concentration of the phosphodiestherase-5 inhibitor vardenafil could provide better protection of vascular grafts against reperfusion injury after long-term cold ischaemic storage. METHODS: Isolated thoracic aorta obtained from rats underwent 24-h cold ischaemic preservation in physiological saline or vardenafil (10(-11) M)-supplemented saline solution. Reperfusion injury was simulated by hypochlorite (200 µM) exposure for 30 minutes. Endothelium-dependent vasorelaxation was assessed, and histopathological and molecular-biological examination of the aortic tissue were performed. RESULTS: Compared with the control group, the saline group showed significantly attenuated endothelium-dependent maximal relaxation (Rmax) to acetylcholine after hypoxia-reoxygenation, which was significantly improved by vardenafil supplementation (Rmax control: 98 ± 1%; saline: 48 ± 6%; vardenafil: 75 ± 4%; p < .05). Vardenafil treatment significantly reduced DNA strand breaks (control: 10.6 ± 6.2%; saline: 72.5 ± 4.0%; vardenafil: 14.2 ± 5.2%; p < .05) and increased cGMP score in the aortic wall (control: 8.2 ± 0.6; saline: 4.5 ± 0.3; vardenafil: 6.7 ± 0.6; p < .05). CONCLUSIONS: Our results support the view that impairment of intracellular cGMP signalling plays a role in the pathogenesis of the endothelial dysfunction induced by cold storage warm reperfusion, which can be effectively reversed by pharmacological phosphodiesterase-5 inhibition.

Decellularized porcine saphenous artery for small-diameter tissue-engineered conduit graft.

Decellularized xenografts have been identified as potential scaffolds for small-diameter vascular substitutes. This study aimed to develop and investigate a biomechanically functional and biocompatible acellular conduit using decellularized porcine saphenous arteries (DPSAs), through a modified decellularization process using Triton X-100/NH4 OH solution and serum-containing medium. Histological and biochemical analysis indicated a high degree of cellular removal and preservation of the extracellular matrix. Bursting pressure tests showed that the DPSAs could withstand a pressure of 1854 ± 164 mm Hg. Assessment of in vitro cell adhesion and biocompatibility showed that porcine pulmonary artery endothelial cells were able to adhere and proliferate on DPSAs in static and rotational culture. After interposition into rabbit carotid arteries in vivo, DPSAs showed patency rates of 60% at 1 month and 50% at 3 months. No aneurysm and intimal hyperplasia were observed in any DPSAs. All patent grafts showed regeneration of vascular elements, and thrombotic occlusion was found to be the main cause of graft failure, probably due to remaining xenoantigens. In conclusion, this study showed the development and evaluation of a decellularization process with the potential to be used as small-diameter grafts.