The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model.

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain's microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Endothelial thrombomodulin downregulation caused by hypoxia contributes to severe infiltration and coagulopathy in COVID-19 patient lungs.

BACKGROUND: Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19. METHODS: We obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy. FINDINGS: The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury. INTERPRETATION: Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2. FUNDING: This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.

The Influence of VE-Cadherin on Adhesion and Incorporation of Breast Cancer Cells into Vascular Endothelium.

During metastasis, cancer cells that originate from the primary tumor circulate in the bloodstream, extravasate, and form micrometastases at distant locations. Several lines of evidence suggest that specific interactions between cancer cells and endothelial cells, in particular tumor cell adhesion to the endothelium and transendothelial migration, play a crucial role in extravasation. Here we have studied the role of vascular endothelial (VE)-cadherin which is expressed aberrantly by breast cancer cells and might promote such interactions. By comparing different human breast cancer cell lines, we observed that the number of cancer cells that adhered to endothelium correlated with VE-cadherin expression levels. VE-cadherin silencing experiments confirmed that VE-cadherin enhances cancer cell adhesion to endothelial cells. However, in contrast, the number of cancer cells that incorporated into the endothelium was not dependent on VE-cadherin. Thus, it appears that cancer cell adhesion and incorporation are distinct processes that are governed by different molecular mechanisms. When cancer cells incorporated into the endothelial monolayer, they formed VE-cadherin positive contacts with endothelial cells. On the other hand, we also observed tumor cells that had displaced endothelial cells, reflecting either different modes of incorporation, or a temporal sequence where cancer cells first form contact with endothelial cells and then displace them to facilitate transmigration. Taken together, these results show that VE-cadherin promotes the adhesion of breast cancer cells to the endothelium and is involved in the initial phase of incorporation, but not their transmigration. Thus, VE-cadherin might be of relevance for therapeutic strategies aiming at preventing the metastatic spread of breast cancer cells.

VDAC1 regulates mitophagy in NLRP3 inflammasome activation in retinal capillary endothelial cells under high-glucose conditions.

Diabetic retinopathy (DR) has been considered to involve mitochondrial alterations and be related to the nucleotide-binding oligomerization domain-like receptors 3 (NLRP3) inflammasome activation. The voltage-dependent anion channel 1 (VDAC1) protein is one of the key proteins that regulates the metabolic and energetic functions of the mitochondria. To explore the involvement of VDAC1 in mitophagy regulation of NLRP3 inflammasome activation under high-glucose (HG) conditions, this study examined expressions of VDAC1, mitochondrial function and mitophagy-related proteins, and NLRP3 inflammasome-related proteins in human retinal capillary endothelial cells (HRCECs) cultured with 30 mM of glucose in the presence or absence of mitophagy inhibitor (Mdivi-1) using Western blot. Mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were detected using flow cytometry. GFP-tagged pAdTrack-VDAC1 adenovirus was used to overexpress VDAC1. Cell biological behaviors, including proliferation, migration, tubule formation, and apoptosis, were also observed. Our results showed that when compared to the normal glucose and high mannitol groups, increased amounts of mitochondrial fragments, reduced mitochondrial membrane potential, increased expression of mitochondrial fission protein Drp 1, decreased expression of mitochondrial fusion protein Mfn 2, accumulation of mtROS, and activation of the NLRP3 inflammasome were observed in the HG group. Meanwhile, HG markedly reduced the protein expressions of PINK1, Parkin and VDAC1. Inhibition of mitophagy reduced PINK1 expression, enhanced NLRP3 expression, but failed to alter VDAC1. VDAC1 overexpression promoted PINK1 expression, inhibited NLRP3 activation and changed the cell biological behaviors under HG conditions. These findings demonstrate that VDAC1-mediated mitophagy plays a crucial role in regulating NLRP3 inflammasome activation in retinal capillary endothelial cells under HG conditions, suggesting that VDAC1 may be a potential target for preventing or treating DR.

Histologic, immunohistochemical, and scanning electron microscopic comparison of pre-iridal monocellular and fibrovascular membranes in normal and glaucomatous canine globes.

OBJECTIVES: (i) To evaluate immunohistochemical labeling of pre-iridal monocellular and fibrovascular membranes and (ii) describe the light and scanning electron microscopic (SEM) characteristics of these membranes in glaucomatous and normal/control canine globes. MATERIALS AND METHODS: All globes were evaluated with light microscopy. Immunohistochemical labeling for CD18, Smooth muscle actin (SMA), and CD117 was completed on 40 canine globes with congenital/anterior segment dysgenesis-associated glaucoma (n = 10), primary/goniodysgenesis-associated glaucoma (n = 10), secondary glaucoma (n = 10), and normal/control globes (n = 10). SEM was completed on 10 globes: 5 with monocellular membranes, 3 with fibrovascular membranes, and 2 without a histologically detectable membrane. RESULTS: Monocellular membranes were detected in all normal/control globes with light microscopy and appeared to be morphologically very similar to those in diseased globes. CD18 labeling was detected in 9/10 monocellular membranes in normal/control globes, 15/23 monocellular, and 7/8 fibrovascular membranes in globes with glaucoma. SMA and CD117 labeling was not detected in monocellular membranes of normal/control globes. SMA was expressed in 10/23 monocellular and 7/8 fibrovascular membranes of glaucomatous globes. CD117 was expressed in 7/23 monocellular and 5/8 fibrovascular membranes of glaucomatous globes. SEM of monocellular membranes revealed a continuous sheet of mostly spindle cells and few individual round cells that extended over the anterior iris face in normal/control and all glaucomatous globes. CONCLUSION: Pre-iridal monocellular membranes are a normal component of the anterior iris surface, and CD18 immunoreactivity suggests some cells within these are of leukocytic origin. SMA and CD117 labeling of monocellular membranes in glaucomatous, but not normal/control globes, suggest metaplastic cellular change secondary to intraocular pathology related to glaucoma.

High intraluminal pressure promotes vascular inflammation via caveolin-1.

The aetiology and progression of hypertension involves various endogenous systems, such as the renin angiotensin system, the sympathetic nervous system, and endothelial dysfunction. Recent data suggest that vascular inflammation may also play a key role in the pathogenesis of hypertension. This study sought to determine whether high intraluminal pressure results in vascular inflammation. Leukocyte adhesion was assessed in rat carotid arteries exposed to 1 h of high intraluminal pressure. The effect of intraluminal pressure on signaling mechanisms including reactive oxygen species production (ROS), arginase expression, and NFĸB translocation was monitored. 1 h exposure to high intraluminal pressure (120 mmHg) resulted in increased leukocyte adhesion and inflammatory gene expression in rat carotid arteries. High intraluminal pressure also resulted in a downstream signaling cascade of ROS production, arginase expression, and NFĸB translocation. This process was found to be angiotensin II-independent and mediated by the mechanosensor caveolae, as caveolin-1 (Cav1)-deficient endothelial cells and mice were protected from pressure-induced vascular inflammatory signaling and leukocyte adhesion. Cav1 deficiency also resulted in a reduction in pressure-induced glomerular macrophage infiltration in vivo. These findings demonstrate Cav1 is an important mechanosensor in pressure-induced vascular and renal inflammation.

Endothelial glycocalyx damage in kidney disease correlates with uraemic toxins and endothelial dysfunction.

BACKGROUND: Damage to the endothelial glycocalyx is an early indicator of vascular damage and a potential marker of endothelial dysfunction. This study aimed to assess the relationship between markers of glycocalyx damage, endothelial dysfunction, and uraemic toxins in patients with chronic kidney disease. METHODS: Healthy controls, CKD patients, dialysis patients, and kidney transplant recipients had biochemical markers of glycocalyx damage (syndecan-1 and hyaluronan), endothelial dysfunction (von Willebrand factor; vWF and vascular cell adhesion molecule; VCAM-1), and uraemic toxins (indoxyl sulphate and p-cresyl sulphate) measured. In addition, Sidestream Darkfield imaging was performed using the novel GlycoCheck™ device to measure glycocalyx width by the perfused boundary region (PBR) in the sublingual microcirculation. RESULTS: Serum markers of glycocalyx damage were highest in the dialysis group (n = 33), followed by CKD patients (n = 32) and kidney transplant recipients (n = 30) compared to controls (n = 30): hyaluronan: 137 (16-1414), 79 (11-257), 57 (14-218) and 23 (8-116) ng/mL, respectively, p < 0.0001; syndecan-1: 81 (40-529), 46 (21-134), 39 (23-72), and 30 (12-138) ng/mL, respectively, p < 0.0001. Markers of endothelial dysfunction followed a similar pattern. No difference in the width of the PBR was detected between these groups (2.01 ± 0.35, 2.07 ± 0.27, 2.06 ± 0.28, and 2.05 ± 0.3 µm, respectively, p = 0.89). Glycocalyx damage correlated with markers of endothelial dysfunction (log-hyaluronan and log-VCAM-1: r = 0.64, p < 0.001) and levels of uraemic toxins (log-hyaluronan and log-indoxyl sulphate: r = 0.48, p < 0.001). CONCLUSIONS: Levels of biochemical markers of glycocalyx and endothelial cell damage are highest in patients receiving dialysis. Glycocalyx and endothelial damage markers correlated with each other, and with uraemic toxins. Although we could not demonstrate a change in PBR, the biochemical markers suggest that glycocalyx damage is most marked in patients with higher levels of uraemic toxins.

Evidence of vascular endothelial dysfunction in Wooden Breast disorder in chickens: Insights through gene expression analysis, ultra-structural evaluation and supervised machine learning methods.

Several gene expression studies have been previously conducted to characterize molecular basis of Wooden Breast myopathy in commercial broiler chickens. These studies have generally used a limited sample size and relied on a binary disease outcome (unaffected or affected by Wooden Breast), which are appropriate for an initial investigation. However, to identify biomarkers of disease severity and development, it is necessary to use a large number of samples with a varying degree of disease severity. Therefore, in this study, we assayed a relatively large number of samples (n = 96) harvested from the pectoralis major muscle of unaffected (U), partially affected (P) and markedly affected (A) chickens. Gene expression analysis was conducted using the nCounter MAX Analysis System and data were analyzed using four different supervised machine-learning methods, including support vector machines (SVM), random forests (RF), elastic net logistic regression (ENET) and Lasso logistic regression (LASSO). The SVM method achieved the highest prediction accuracy for both three-class (U, P and A) and two-class (U and P+A) classifications with 94% prediction accuracy for two-class classification and 85% for three-class classification. The results also identified biomarkers of Wooden Breast severity and development. Additionally, gene expression analysis and ultrastructural evaluations provided evidence of vascular endothelial cell dysfunction in the early pathogenesis of Wooden Breast.

Endothelial cell disruption drives increased blood-brain barrier permeability and cerebral edema in the Dahl SS/jr rat model of superimposed preeclampsia.

Preeclampsia is characterized by increases in blood pressure and proteinuria in late pregnancy, and neurological symptoms can appear in the form of headaches, blurred vision, cerebral edema, and, in the most severe cases, seizures (eclampsia). The causes for these cerebral manifestations remain unknown, so the use of animal models that mimic preeclampsia is essential to understanding its pathogenesis. The Dahl salt-sensitive (Dahl SS/jr) rat model develops spontaneous preeclampsia superimposed on chronic hypertension; therefore, we hypothesized that the Dahl SS/jr rat would display cerebrovascular features similar to those seen in human preeclampsia. Furthermore, we predicted that this model would allow for the identification of mechanisms underlying these changes. The pregnant Dahl SS/jr rat displayed increased cerebral edema and blood-brain barrier disruption despite tighter control of cerebral blood flow autoregulation and vascular smooth muscle myogenic tone. Analysis of cerebral endothelial cell morphology revealed increased opening of tight junctions, basement membrane dissolution, and vesicle formation. RNAseq analysis identified that genes related to endothelial cell tight junctions and blood-brain barrier integrity were differentially expressed in cerebral vessels from pregnant Dahl SS/jr compared with healthy pregnant Sprague Dawley rats. Overall, our data reveal new insights into mechanisms involved in the cerebrovascular dysfunction of preeclampsia.NEW & NOTEWORTHY This study uses the Dahl SS/jr rat as a preclinical model of spontaneous superimposed preeclampsia to demonstrate uncoupling of cerebral vascular permeability and blood-brain barrier disruption from cerebral blood flow autoregulatory dysfunction and myogenic tone. Additionally, the data presented in this study lay the foundational framework on which future experiments assessing specific transcellular transport components such as individual transporter protein expression and components of the vesicular transport system (caveolae) can be built to help reveal a potential direct mechanistic insight into the causes of cerebrovascular complications during preeclamptic pregnancies.

Preserving the endothelium in saphenous vein graft with both conventional and no-touch preparation.

BACKGROUND: Despite the inferior patency compared to arterial grafts, a saphenous vein graft (SVG) is widely used for coronary artery bypass grafting (CABG). A lower atherosclerosis rate and higher patency have been reported for SVG obtained via the no-touch technique (NT) than via conventional preparation (CV). Although CV-mediated endothelial dysfunction is implied, the precise mechanism underlying the higher patency with NT is poorly understood. METHODS: Human residual SVGs during CABG and SVG sections after autopsy were analyzed. The endothelial surface was observed using scanning electron microscopy (SEM) and blindly compared between CV and NT. The endothelial integrity was also analyzed with immunohistochemistry. RESULTS: Unexpectedly, the hyperfine structure on SEM was comparable between CV and NT before grafting, and microvillus, a characteristic of endothelium, was indistinguishable between them. Von Willebrand Factor, an endothelial marker, was equally detected throughout the vascular wall in both groups from residual and postmortem sections. CONCLUSIONS: The morphological integrity of the endothelium was successfully preserved in SVG with CV, even at an ultrastructural level. Although its functionality remains to be addressed, other factors than the endothelium may be involved in the high patency obtained by NT. The present findings suggest that the characteristics of NT and surgical methodology should be reconsidered.

Aspirin restores endothelial function by mitigating 17ß-estradiol-induced α-SMA accumulation and autophagy inhibition via Vps15 scaffold regulation of Beclin-1 phosphorylation.

AIMS: Previous studies have shown that the widespread use of estrogen preparations can cause adverse outcomes such as thrombosis and cardiovascular disease. Autophagy is a biochemical process necessary to maintain cell homeostasis. The present study investigated whether E-2 mediates autophagy-induced endothelial cell dysfunction. The role of aspirin in this process was then studied. MAIN METHODS: Western blot, fluorescence microscopy, electron transmission microscopy, plasma construction and transfection, vasoreactivity study in wire myograph are all used in this study. KEY FINDINGS: We found that E-2 activated the PI3K/mTOR signaling pathway and inhibited the formation of the Atg14L-Beclin1-Vps34-Vps15 complex, thereby inhibiting autophagy. Aspirin promoted Beclin1 phosphorylation in autophagy initiation complexes and enhanced autophagy. Furthermore, E-2 treatment of HAECs resulted in endothelial dysfunction by inhibiting autophagy and leading to accumulation of α-smooth muscle actin (α-SMA). E-2 inhibited the activation of eNOS and reduced the expression of eNOS protein. In the mouse aortic vascular function test, E-2 disrupted endothelium-dependent vasodilation. An α-SMA-shRNA lentivirus eliminated the disruption to endothelium-dependent vasodilation by E-2. Aspirin inhibited α-SMA accumulation by enhancing autophagy, reversed endothelial functional impairment caused by E-2, and promoted endothelium-dependent vasodilation. SIGNIFICANCE: This study provides new evidence that E-2 inhibits autophagy and induces abnormal accumulation of α-SMA, resulting in endothelial cell dysfunction and affecting vasodilation. Aspirin can effectively restore the endothelial cell function disrupted E-2.

Role of Extracellular Vesicles in Pulmonary Arterial Hypertension: Modulation of Pulmonary Endothelial Function and Angiogenesis.

OBJECTIVE: Extracellular vesicles (EVs) have the potential to act as intercellular communicators. The aims were to characterize circulating EVs in patients with pulmonary arterial hypertension (PAH) and to explore whether these EVs contribute to endothelial activation and angiogenesis. Approach and Results: Patients with PAH (n=70) and healthy controls (HC; n=20) were included in this cross-sectional study. EVs were characterized and human pulmonary endothelial cells (hPAECs) were incubated with purified EVs. Endothelial cell activity and proangiogenic markers were analyzed. Tube formation analysis was performed for hPAECs, and the involvement of PSGL-1 (P-selectin glycoprotein ligand 1) was evaluated. The numbers of CD62P+, CD144+, and CD235a EVs were higher in blood from PAH compared with HC. Thirteen proteins were differently expressed in PAH and HC EVs, where complement fragment C1q was the most significantly elevated protein (P=0.0009) in PAH EVs. Upon EVs-internalization in hPAECs, more PAH compared with HC EVs evaded lysosomes (P<0.01). As oppose to HC, PAH EVs stimulated hPAEC activation and induced transcription and translation of VEGF-A (vascular endothelial growth factor A; P<0.05) and FGF (fibroblast growth factor; P<0.005) which were released in the cell supernatant. These proangiogenic proteins were higher in patient with PAH plasma compered with HC. PAH EVs induced a complex network of angiotubes in vitro, which was abolished by inhibitory PSGL-1antibody. Anti-PSGL-1 also inhibited EV-induced endothelial cell activation and PAH EV dependent increase of VEGF-A. CONCLUSIONS: Patients with PAH have higher levels of EVs harboring increased amounts of angiogenic proteins, which induce activation of hPAECs and in vitro angiogenesis. These effects were partly because of platelet-derived EVs evasion of lysosomes upon internalization within hPAEC and through possible involvement of P-selectin-PSGL-1 pathway.

Angiotensin-(1-7) Treatment Restores Pancreatic Microcirculation Profiles: A New Story in Acute Pancreatitis.

OBJECTIVES: The aim of this study was to investigate the changes of pancreatic microvascular vasomotion and blood distribution pattern in acute pancreatitis (AP), and whether Angiotensin (Ang)-(1-7) treatment could restore pancreatic microcirculation profiles. METHODS: Mice were randomly separated into control, AP, and Ang-(1-7)-treated AP (A-AP) group. Acute pancreatitis was induced in mice by intraperitoneal injection of cerulein and lipopolysaccharide. Pancreatitis was confirmed by histopathology, serum amylase, and high-sensitive C-reactive protein. Pancreatic microvascular vasomotion and blood distribution pattern in AP progression were assessed by laser Doppler. Meanwhile, ultrastructural changes of pancreatic microcirculation, including microvascular cavity and wall and endothelial mitochondria, were evaluated by transmission electron microscopy. RESULTS: Acute pancreatitis mice exhibited pathological pancreatic injuries with lower blood distribution pattern and decreased average blood perfusion, relative velocity, effective frequency, and amplitude of microvascular vasomotion. The pancreatic pathological injuries in Ang-(1-7)-treated mice were significantly alleviated. Consistently, Ang-(1-7) treatment led to a restoration in pancreatic microcirculation profiles. Furthermore, non-Ang-(1-7)-treated mice showed an irregular microvascular wall, narrow cavity, and swelling mitochondria, and these ultrastructural impairments were reversed by Ang-(1-7) administration. CONCLUSIONS: Pancreatic microcirculation profiles are abnormal in the progression of AP. Angiotensin-(1-7) administration could restore functional status of pancreatic microcirculation.

Cav-1 (Caveolin-1) Deficiency Increases Autophagy in the Endothelium and Attenuates Vascular Inflammation and Atherosclerosis.

OBJECTIVE: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1-deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr-/- and Cav-1-/-Ldlr-/- mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1-/- mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis. CONCLUSIONS: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1-deficient mice attenuates the atheroprotection observed in Cav-1-/- mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.

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.

Effect of resveratrol combined with atorvastatin on re-endothelialization after drug-eluting stents implantation and the underlying mechanism.

AIMS: To explore whether the combination of atorvastatins and resveratrol is superior to each individual drug alone regarding re-endothelialization after drug-eluting stents (DESs) implantation. MATERIALS AND METHODS: Ninety-four rabbits were randomized into control, atorvastatin, resveratrol, and combined medication groups. Abdominal aorta injury was induced via ballooning, followed by DES implantation. Neointimal formation and re-endothelialization after stent implantation were assessed via optical coherence tomography and scanning electron microscopy. The effects of resveratrol and atorvastatin on bone marrow-derived mesenchymal derived stem cells (BMSCs) were assessed. KEY FINDINGS: Compared with the findings in the resveratrol and atorvastatin groups, the neointimal area and mean neointimal thickness were greater in the combined medication group, which also exhibited improved re-endothelialization. Compared with the effects of monotherapy, combined treatment further protected BMSCs against rapamycin-induced apoptosis and improved cell migration. Combined medication significantly upregulated Akt, p-Akt, eNOS, p-eNOS, and CXCR4 expression in BMSCs compared with the effects of monotherapy, and these effects were abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. SIGNIFICANCE: The combination of atorvastatin and resveratrol has the potential of accelerating re-endothelialization after stent implantation, reducing the risk of thrombosis and improving the safety of DESs.

Interference With ESAM (Endothelial Cell-Selective Adhesion Molecule) Plus Vascular Endothelial-Cadherin Causes Immediate Lethality and Lung-Specific Blood Coagulation.

OBJECTIVE: Vascular endothelial (VE)-cadherin is of dominant importance for the formation and stability of endothelial junctions, yet induced gene inactivation enhances vascular permeability in the lung but does not cause junction rupture. This study aims at identifying the junctional adhesion molecule, which is responsible for preventing endothelial junction rupture in the pulmonary vasculature in the absence of VE-cadherin. Approach and Results: We have compared the relevance of ESAM (endothelial cell-selective adhesion molecule), JAM (junctional adhesion molecule)-A, PECAM (platelet endothelial cell adhesion molecule)-1, and VE-cadherin for vascular barrier integrity in various mouse tissues. Gene inactivation of ESAM enhanced vascular permeability in the lung but not in the heart, skin, and brain. In contrast, deletion of JAM-A or PECAM-1 did not affect barrier integrity in any of these organs. Blocking VE-cadherin with antibodies caused lethality in ESAM-/- mice within 30 minutes but had no such effect in JAM-A-/-, PECAM-1-/- or wild-type mice. Likewise, induced gene inactivation of VE-cadherin caused rapid lethality only in the absence of ESAM. Ultrastructural analysis revealed that only combined interference with VE-cadherin and ESAM disrupted endothelial junctions and caused massive blood coagulation in the lung. Mechanistically, we could exclude a role of platelet ESAM in coagulation, changes in the expression of other junctional proteins or a contribution of cytoplasmic signaling domains of ESAM. CONCLUSIONS: Despite well-documented roles of JAM-A and PECAM-1 for the regulation of endothelial junctions, only for ESAM, we detected an essential role for endothelial barrier integrity in a tissue-specific way. In addition, we found that it is ESAM which prevents endothelial junction rupture in the lung when VE-cadherin is absent.

Sildenafil reduces aortic endothelial dysfunction and structural damage in spontaneously hypertensive rats: Role of NO, NADPH and COX-1 pathways.

Arterial hypertension is a condition associated with endothelial dysfunction, accompanied by an imbalance in the production of reactive oxygen species (ROS) and NO. The aim of this study was to investigate and elucidate the possible mechanisms of sildenafil, a selective phosphodiesterase-5 inhibitor, actions on endothelial function in aortas from spontaneously hypertensive rats (SHR). SHR treated with sildenafil (40 mg/kg/day, p.o., 3 weeks) were compared to untreated SHR and Wistar-Kyoto (WKY) rats. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and vascular reactivity was determined in isolated rat aortic rings. Circulating endothelial progenitor cells and systemic ROS were measured by flow cytometry. Plasmatic total antioxidant capacity, NO production and aorta lipid peroxidation were determined by spectrophotometry. Scanning electron microscopy was used for structural analysis of the endothelial surface. Sildenafil reduced high SBP and partially restored the vasodilator response to acetylcholine and sodium nitroprusside in SHR aortic rings. Using selective inhibitors, our experiments revealed an augmented participation of NO, with a simultaneous decrease of oxidative stress and of cyclooxygenase-1 (COX-1)-derived prostanoids contribution in the endothelium-dependent vasodilation in sildenafil-treated SHR compared to non-treated SHR. Also, the relaxant responses to sildenafil and 8-Br-cGMP were normalized in sildenafil-treated SHR and sildenafil restored the pro-oxidant/antioxidant balance and the endothelial architecture. In conclusion, sildenafil reverses endothelial dysfunction in SHR by improving vascular relaxation to acetylcholine with increased NO bioavailability, reducing the oxidative stress and COX-1 prostanoids, and improving cGMP/PKG signaling. Also, sildenafil reduces structural endothelial damage. Thus, sildenafil is a promising novel pharmacologic strategy to treat endothelial dysfunction in hypertensive states reinforcing its potential role as adjuvant in the pharmacotherapy of cardiovascular diseases.

The effect of different flushing methods in a short peripheral catheter.

PURPOSE: To develop a rabbit model of a short peripheral catheter (SPC) and to observe the effects of different flushing methods on blood vessels. METHODS: Thirty rabbits were randomly divided into three groups (A, B, and C), with ten rabbits per group. In group A, we used pulsed flush; in group B, we used uniform flush; and no treatment was used in group C. RESULTS: We observed that a uniform flush reduced blockage, phlebitis, and exudation compared to a pulsed flush by visual observation. The histopathological examination found that the morphological changes in group A were more severe than in group B and C related to loss of venous endothelial cells, inflammatory cell infiltration, edema, epidermal and chondrocyte degeneration, except for the thrombosis on group B that was more serious than in group A, especially in the distal side of puncture points. The distal region of groups A and B had more inflammatory cell infiltration than the proximal region. Thrombosis was more severe in the distal region than in the proximal region in group B. CONCLUSIONS: The uniform flush produced less damage to the vascular endothelium and surrounding tissues and was superior to the pulsed flush. However, the uniform flush is prone to thrombosis.

Effect of the Hemoxygenase-Carbon Monoxide (HO-CO) System on the Reactivity of Uterine Artery Branches in Rats.

First to fourth-order branches of the uterine artery in sexually mature female Wistar rats were studied by biomicroscopy. After administration of a CO donor hemin (60 mM), the diameters of large uterine branches with a well-developed muscle layer markedly increased, while the increase in diameter of small vessels with one often interrupted layer of smooth muscle cells increased insignificantly. Zinc protoporphyrin IX (30 mM) in all cases blocked this effect. However, zinc protoporphyrin IX does not affect NO-mediated reaction of the branches of the uterine artery caused by administration of L-arginine (60 mM), and L-NAME did not significantly affect reactivity of uterine artery branches associated with the hemoxygenase-CO system. In contrast to NO, CO produced less potent and rapid, but more sustained effect. The target for the hemoxygenase-CO system is mainly arteries with developed muscular layer, while the target for the NO synthase-NO is small vessels where endothelium plays a Rdecisive role in the regulation of vasomotor reactions.