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.

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.

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.

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.

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.

Reestablishment of the endothelial lining by endothelial cell therapy stabilizes experimental abdominal aortic aneurysms.

BACKGROUND: Loss of the endothelium and its replacement by a thick thrombus are structural features of human abdominal aortic aneurysms (AAAs). In AAAs, the relationship between aortic diameter expansion, the presence of thrombus, and the lack of endothelial cells (ECs) remains unexplored. We hypothesized that reendothelialization by cell therapy would modulate aortic wall destruction and ultimately stabilize AAAs. We evaluated the impact of local seeding of rat aortic ECs or peripheral blood-derived outgrowth ECs on AAA evolution. METHODS AND RESULTS: Rat aortic ECs (n=30) or serum-free medium (controls; n=29) were seeded endovascularly immediately (day 0) or 14 days after surgery in the rat xenograft model. Rat aortic EC seeding prevented AAA formation and stabilized formed AAAs at 28 days (diameter increase at day 0+28, 51±6% versus 83±6%; day 14+28, -1±4% versus 22±6% in rat aortic ECs and controls, respectively; P<0.01). This stabilizing effect was associated with the reestablishment of the endothelial lining, the suspension of proteolysis, and the reconstitution of new aortic wall rich in smooth muscle cells and extracellular matrix. Transplanted rat aortic ECs did not participate directly in aortic wall repair but exerted their healing properties through paracrine mechanisms involving the upregulation of endothelium-derived stabilizing factors and the recruitment of resident vascular cells. In rats, the transplantation of outgrowth ECs (n=7) significantly reduced by 30% the progression of AAAs and restored the abluminal endothelium at 28 days compared with controls (n=9). CONCLUSION: Our study demonstrates the potential of restoring the endothelial lining to control AAA dynamics and designates ECs as an efficient therapy to stop AAA expansion.

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.

Enhanced angiogenic and cardiomyocyte differentiation capacity of epigenetically reprogrammed mouse and human endothelial progenitor cells augments their efficacy for ischemic myocardial repair.

RATIONALE: Although bone marrow endothelial progenitor cell (EPC)-based therapies improve the symptoms in patients with ischemic heart disease, their limited plasticity and decreased function in patients with existing heart disease limit the full benefit of EPC therapy for cardiac regenerative medicine. OBJECTIVE: We hypothesized that reprogramming mouse or human EPCs, or both, using small molecules targeting key epigenetic repressive marks would lead to a global increase in active gene transcription, induce their cardiomyogenic potential, and enhance their inherent angiogenic potential. METHOD AND RESULTS: Mouse Lin-Sca1(+)CD31(+) EPCs and human CD34(+) cells were treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid), and G9a histone dimethyltransferase. A 48-hour treatment led to global increase in active transcriptome, including the reactivation of pluripotency-associated and cardiomyocyte-specific mRNA expression, whereas endothelial cell-specific genes were significantly upregulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into cardiomyocytes. Treatment with epigenetic-modifying agents show marked increase in histone acetylation on cardiomyocyte and pluripotent cell-specific gene promoters. Intramyocardial transplantation of reprogrammed mouse and human EPCs in an acute myocardial infarction mouse model showed significant improvement in ventricular functions, which was histologically supported by their de novo cardiomyocyte differentiation and increased capillary density and reduced fibrosis. Importantly, cell transplantation was safe and did not form teratomas. CONCLUSIONS: Taken together, our results suggest that epigenetically reprogrammed EPCs display a safe, more plastic phenotype and improve postinfarct cardiac repair by both neocardiomyogenesis and neovascularization.

Microfluidic single-cell analysis shows that porcine induced pluripotent stem cell-derived endothelial cells improve myocardial function by paracrine activation.

RATIONALE: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models. OBJECTIVE: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia. METHODS AND RESULTS: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery. CONCLUSIONS: In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

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.

Preservation of endothelial vascular function of saphenous vein grafts after long-time storage with a recently developed potassium-chloride and N-acetylhistidine enriched storage solution.

OBJECTIVES: Saphenous vein grafts are still commonly used in cardiac, vessel and also in transplant surgery. In cardiac surgery, a desperate graft situation could force to keep nonutilized explanted vein segments after CABG in reserve for case of early graft failure. Historically there were no options for adequate long-time graft storage protecting the endothelial layer with its important antithrombotic and immunosuppressive functional aspects. Commonly isotonic saline solution (sodium chloride [NaCl]) has been used as a storing solution in this case. We investigated the impact of long-time storage in NaCl and a recently developed potassium-chloride and N-acetylhistidine enriched storage solution (TiProtec, Dr. Köhler Chemie, Germany) on endothelial function of saphenous veins. METHODS: Saphenous vein segments (n = 19) were intraoperatively isolated and stored for 24 and 96 hours. The segments were examined in a Mulvany-myograph to assess vessel function. Following preconstriction with norepinephrine, dose-response curves were assessed for relaxation with bradykinin and sodium-nitroprusside. We compared developed maximum wall tension and endothelial cell and smooth muscle cell (SMC) dependent vasodilatory function. RESULTS: Maximum vessel wall tension was significantly better preserved in TiProtec-stored vessels after 24 h in comparison to segments stored in NaCl (5.11 ± 4.79 mN/mm vs. 2.48 ± 2.43 mN/mm; p = 0.033) and 96 h (4.94 ± 2.82 mN/mm vs. 2.80 ± 1.76 mN/mm; p = 0.042). Likewise endothelium-derived vasodilatory function was maintained significantly after 24 hours in TiProtec-stored vessels (36.9 ± 2.6% vs. 11.8 ± 30.9%; p = 0.005). After 96 hours, endothelium-dependent vascular function was nearly abolished in NaCl-stored vessels, but largely preserved in TiProtec-stored segments (20.6 ± 2.9% vs. 1.9 ± 4.3% in NaCl; p = 0.015). Sodium nitroprusside-mediated SMC-vasodilatory function was better maintained after 24 hours of storage in TiProtec group (88.8 ± 6.4% vs. 61.3 ± 8.2%; p = 0.009). After 96 hours of storage, SMC relaxation did not significantly differ between both storage groups which might be due to a distinct reduction of contractile function in NaCl-stored vessels (98.6 ± 5.0% and 77.9 ± 10.5% for Tiprotec and NaCl, respectively). CONCLUSION: Vessel functions comprising contraction, endothelium-dependent and -independent vasodilatation are significantly reduced following 24 hours of cold storage in NaCl. After 96 hours of storage in NaCl these functions are nearly totally abolished. TiProtec is able to largely reduce this loss of function during cold storage. Therefore, TiProtec is a feasible option for longer term storage of saphenous vein grafts in CABG vessel and transplant surgery.

Microparticle release in remote ischemic conditioning mechanism.

Remote ischemic conditioning (RCond) induced by short periods of ischemia and reperfusion of an organ or tissue before myocardial reperfusion is an attractive strategy of cardioprotection in the context of acute myocardial infarction. Nonetheless, its mechanism remains unknown. A humoral factor appears to be involved, although its identity is currently unknown. We hypothesized that the circulating microparticles (MPs) are the link between the remote tissue and the heart. MPs from rats and healthy humans undergoing RCond were characterized. In rats, RCond was induced by 10 min of limb ischemia. In humans, RCond was induced by three cycles of 5-min inflation and 5-min deflation of a blood-pressure cuff. In the second part of the study, rats underwent 40 min myocardial ischemia followed by 2 h reperfusion. Infarct size was measured and compared among three groups of rats: 1) myocardial infarction alone (MI) (n = 6); 2) MI + RCond started 20 min after coronary ligation (n = 6); and 3) MI + injection of RCond-derived rat MPs (MI + MPs) (n = 5). MPs from endothelial cells (CD54(+) and CD146(+) for rats and humans, respectively) and procoagulant MPs (Annexin V(+)) markedly increased after RCond, both in rats and humans. RCond reduced infarct size (24.4 ± 5.9% in MI + RCond vs. 54.6 ± 4.7% in MI alone; P < 0.01). Infarct size did not decrease in MI + MPs compared with MI alone (50.2 ± 6.4% vs. 54.6 ± 4.7%, not significantly different). RCond increased endothelium-derived and procoagulant MPs in both rats and humans. However, MP release did not appear to be a biological vector of RCond in our model.

CD146 short isoform increases the proangiogenic potential of endothelial progenitor cells in vitro and in vivo.

RATIONALE: CD146, a transmembrane immunoglobulin mainly expressed at the intercellular junction of endothelial cells, is involved in cell-cell cohesion, paracellular permeability, monocyte transmigration and angiogenesis. CD146 exists as 2 isoforms, short (sh) and long (lg), but which isoform is involved remains undefined. OBJECTIVE: The recently described role of CD146 in angiogenesis prompted us to investigate which isoform was involved in this process in human late endothelial progenitors (EPCs), with the objective of increasing their proangiogenic potential. METHODS AND RESULTS: Immunofluorescence experiments showed that, in subconfluent EPCs, shCD146 was localized in the nucleus and at the migrating edges of the membrane, whereas lgCD146 was intracellular. In confluent cells, shCD146 was redistributed at the apical membrane and lgCD146 was directed toward the junction. In contrast to lgCD146, shCD146 was overexpressed in EPCs as compared to mature endothelial cells and upregulated by vascular endothelial growth factor and SDF-1 (stromal cell-derived factor 1). Study of the properties of both isoforms in vitro provided evidence that shCD146 was involved in EPC adhesion to activated endothelium, migration, and proliferation, with a paracrine secretion of interleukin-8 or angiopoietin 2, whereas lgCD146 was implicated in stabilization of capillary-like structures in Matrigel and transendothelial permeability. In an animal model of hindlimb ischemia, transplantation of shCD146-modified EPCs selectively promoted both EPC engraftment and blood flow. CONCLUSIONS: Altogether, these findings establish that CD146 isoforms display distinct functions in vessels regeneration. Selective improvement of therapeutic angiogenesis by shCD146 overexpression suggests a potential interest of shCD146-transduced EPCs for the treatment of peripheral ischemic disease.

Endothelial cells derived from human iPSCS increase capillary density and improve perfusion in a mouse model of peripheral arterial disease.

OBJECTIVE: Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease. METHODS AND RESULTS: Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and vascular endothelial growth factor. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144, and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging and green fluorescence protein for fluorescent detection. The hiPSC-ECs (5×10(5)) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). Bioluminescence imaging showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 versus 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284±155 versus 797±206 capillaries/mm(2)) (P<0.002). CONCLUSION: This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.

Magnetic resonance imaging targeting of intracranial glioma xenografts by Resovist-labeled endothelial progenitor cells.

Accumulating evidence suggests that endothelial progenitor cells (EPCs) play a key role in the development and infiltration of gliomas. Thus, it has been considered that EPCs may be good vehicles for delivering anti-angiogenesis genes for tumor therapy. However, limited means of tracking these cells in vivo has restricted the effective evaluation of the curative effects of genetically modified EPCs in gliomas at different stages. The aim of this study was to develop a non-invasive method to monitor the migration of EPCs to gliomas using 1.5-T MR scanning. We successfully labeled EPCs isolated from cord blood with Resovist-PLL without any influence on the biological properties of these cells. After intravenous administration into glioma-bearing nude mice, the labeled EPCs specifically homed to gliomas and could be reliably tracked by 1.5-T MR as early as 1 day after transplantation, causing a signal loss on T2-weighted images. The dark area was detected throughout the entire tumor zone on day 5, and did not develop a ring as previously described. Histological analysis showed the labeled cells were mainly located at the periphery of the tumor where abundant neo-vessels were identified using CD34 staining; this finding indicates that the transplanted cells may be able to differentiate into ECs and become incorporated into glioma neovasculature. These results suggested that Resovist labeling of EPCs is feasible, efficient and safe for MRI tracking, and 1.5-T MR could be a powerful method for in vivo monitoring of EPCs as an anti-angiogenic drug therapy vector targets against glioma.

Prelining autogenic endothelial cells in allogeneic vessels inhibits thrombosis and intimal hyperplasia: an efficacy study in dogs.

BACKGROUND: The long-term patency rates in vascular transplants (diameter<3.0-4.0mm) are very low due to thrombus formation and intimal hyperplasia. A possible mechanism is the loss of the endothelial cells (ECs) lining. Previous attempts to reseed ECs had poor results due to seeded cell loss, severe antigenicity, and low compliance. The objectives of this study were to generate an allogeneic vascular substitution with autogenic ECs and low antigenicity. METHODS: ECs from mongrels were obtained and multiplied in vitro, then seeded to the allogeneic vein luminal surface, which was preserved by freeze-drying radiation. The cultivated cells' secretory function was confirmed by von Willebrand factor detection. The allogeneic vascular was then transplanted into animals' necks in situ. The physical properties, EC state, and vascular structure of the allogeneic vascular grafts were studied. RESULTS: The secretory function of ECs did not vary in vitro. The expression level of MHC-II antigen in freeze-dried radiation-treated vasculature was lower than normal fresh vasculature (P<0.05). ECs covered the vascular inner surface and adhered tightly after implantation. As assessed by scanning electron micrograph, most ECs adhered tightly, and the cell polarity changed in accordance with the direction of the force. Allograft blood vessels with autogenic ECs implanted showed significant decreases in both thrombosis and intimal hyperplasia. CONCLUSION: Allograft blood vessels seeded with autogenic ECs improved the patency of small-diameter grafts in a canine model. Our study showed a significant decrease in both thrombosis and intimal hyperplasia.

Is endothelium the origin of endothelial progenitor cells?

Endothelial cells provide the dynamic lining of blood vessels throughout the body and provide many tissue-specific functions, in addition to providing a nonthrombogenic surface for blood cells and conduit for oxygen and nutrient delivery. As might be expected, some endothelial cells are injured or become senescent and are sloughed into the bloodstream, and most circulating endothelial cells display evidence of undergoing apoptosis or necrosis. However, there are rare viable circulating endothelial cells that display properties consistent with those of a progenitor cell for the endothelial lineage. This article reviews historical and current literature to present some evidence that the endothelial lining of blood vessels may serve as a source for rare endothelial colony-forming cells that display clonal proliferative potential, self-renewal, and in vivo vessel forming ability. The article also discusses the current gaps in our knowledge to prove whether the colony-forming cells are in fact derived from vascular endothelium.

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.

Differences in donor CXCR4 expression levels are correlated with functional capacity and therapeutic outcome of angiogenic treatment with endothelial colony forming cells.

CXCR4 expression is important for cell migration and recruitment, suggesting that the expression levels of CXCR4 may be correlated with functional activity of implanted cells for therapeutic neovascularization. Here, we examined differences between umbilical cord blood (CB) donors in the CXCR4 levels of endothelial colony forming cells (ECFCs), which are a subtype of endothelial progenitor cells (EPCs). We investigated the relationships between CXCR4 expression level and SDF-1alpha-induced vascular properties in vitro, and their in vivo contributions to neovascularization. We found that ECFCs isolated from different donors showed differences in CXCR4 expression that were linearly correlated with SDF-1alpha-induced migratory capacity. ECFCs with high CXCR4 expression showed enhanced ERK and Akt activation in response to SDF-1alpha. In addition, SDF-1alpha-induced migration and ERK1/2, Akt, and eNOS activation were reduced by AMD3100, a CXCR4-specific peptide antagonist, or by siRNA-CXCR4. Administration of high-CXCR4-expressing ECFCs resulted in a significant increase in therapeutic potential for blood flow recovery, tissue healing and capillary density compared to low-CXCR4-expressing ECFCs in hindlimb ischemia. Taken together, the functional differences among ECFCs derived from different donors depended on the level of CXCR4 expression, suggesting that CXCR4 expression levels in ECFCs could be a predictive marker for success of ECFC-based angiogenic therapy.