Surface Of Vascular Endothelium Research Articles

Application of CD34 expression combined with three-phase dynamic contrast-enhanced computed tomography scanning in preoperative staging of gastric cancer.

Accurate preoperative staging of gastric cancer (GC), a common malignant tumor worldwide, is critical for appropriate treatment plans and prognosis. Dynamic three-phase enhanced computed tomography (CT) scanning for preoperative staging of GC has limitations in evaluating tumor angiogenesis. CD34, a marker on vascular endothelial cell surfaces, is promising in evaluating tumor angiogenesis. We explored the value of their combination for preoperative staging of GC to improve the efficacy and prognosis of patients with GC. To explore the evaluation value of CD34 expression + dynamic three-phase enhanced CT scanning in preoperative staging of GC. Medical records of 106 patients with GC treated at the First People's Hospital of Lianyungang between February 2021 and January 2023 were retrospectively studied. All patients underwent three-phase dynamic contrast-enhanced CT scanning before surgery, and CD34 was detected in gastroscopic biopsy specimens. Using surgical and pathological results as the gold standard, the diagnostic results of three-phase dynamic contrast-enhanced CT scanning at different T and N stages were analyzed, and the expression of CD34-marked microvessel density (MVD) at different T and N stages was determined. The specificity and sensitivity of three-phase dynamic contrast-enhanced CT and CD34 in T and N staging were calculated; those of the combined diagnosis of the two were evaluated in parallel. Independent factors affecting lymph node metastasis were analyzed using multiple logistic regression. The accuracy of three-phase dynamic contrast-enhanced CT scanning in diagnosing stages T1, T2, T3 and T4 were 68.00%, 75.00%, 79.41%, and 73.68%, respectively, and for diagnosing stages N0, N1, N2, and N3 were 75.68%, 74.07%, 85.00%, and 77.27%, respectively. CD34-marked MVD expression increased with increasing T and N stages. Specificity and sensitivity of three-phase dynamic contrast-enhanced CT in T staging were 86.79% and 88.68%; for N staging, 89.06% and 92.86%; for CD34 in T staging, 64.15% and 88.68%; and for CD34 in N staging, 84.38% and 78.57%, respectively. Specificity and sensitivity of joint diagnosis in T staging were 55.68% and 98.72%, and N staging were 75.15% and 98.47%, respectively, with the area under the curve for diagnosis improving accordingly. According to multivariate analysis, a longer tumor diameter, higher pathological T stage, lower differentiation degree, and higher expression of CD34-marked MVD were independent risk factors for lymph node metastasis in patients with GC. With high accuracy in preoperatively determining the invasion depth and lymph node metastasis of GC, CD34 expression and three-phase dynamic contrast-enhanced CT can provide a reliable basis for surgical resection.

Proanthocyanidins: A novel approach to Henoch‑Schonlein purpura through balancing immunity and arresting oxidative stress via TLR4/MyD88/NF‑κB signaling pathway (Review).

Henoch-Schonlein purpura (HSP), a recurrent and immunoglobulin (Ig)A-mediated vasculitis, presents not only as skin lesions but also as systemic involvement that can be life-threatening. Although the etiology of HSP remains unknown, immune imbalance and oxidative stress (OS) are primary contributors to its pathogenesis, alongside the abnormal activation of Toll-like receptor (TLR)/myeloid differentiation primary response gene 88 (MyD88)/nuclear factor-κB (NF-κB) pathway. TLRs, especially TLR4, stimulate downstream signaling molecules such as NF-κB and proinflammatory cytokines, which are released when TLRs combine with the key adapter molecule MyD88. This leads to the activation of T helper (Th) cell 2/Th17 and overproduction of reactive oxygen species (ROS). The function of regulatory T (Treg) cells is suppressed in the process. Th17/Treg imbalance then produces various inflammatory cytokines to promote proliferation and differentiation of B cells and the secretion of antibodies. IgA is secreted, and it binds to vascular endothelial surface receptors where the complex induces injury of the vascular endothelial cells. Additionally, excessive ROS creates OS that leads to an inflammatory response and vascular cell apoptosis or necrosis, thereby contributing to vascular endothelial damage and HSP occurrence. Proanthocyanidins are active compounds naturally enriched in fruits, vegetables and plants. Proanthocyanidins have diverse properties, including anti-inflammatory, antioxidant, antibacterial, immunoregulatory, anticarcinogenic and vascular protective effects. Proanthocyanidins are used in the management of various diseases. Proanthocyanidins regulate T cells, equilibrate immunity and arrest OS by inhibiting the TLR4/MyD88/NF-κB signaling pathway. Considering the pathogenesis of HSP and the properties of proanthocyanidins, the present study hypothesized that these compounds may potentially lead to HSP recovery through modulating the immune equilibrium and preventing OS by inhibiting the TLR4/MyD88/NF-κB pathway. To the best of our knowledge, however, little is known about the positive effects of proanthocyanidins against HSP. The present review summarizes the potential of proanthocyanidins to treat HSP.

MiR-126-Loaded Immunoliposomes against Vascular Endothelial Inflammation In Vitro and Vivo Evaluation.

Due to the accompaniment of vascular endothelial inflammation during the occurrence and development of cardiovascular diseases (CVD), treatment modalities against vascular endothelial inflammation have been intensively investigated for CVD prevention and/or treatment. Vascular cell adhesion molecule-1 (VCAM-1) is a typical transmembrane inflammatory protein specifically expressed by inflammatory vascular endothelial. By inhibiting VCAM-1 expression through the miR-126 mediated pathway, vascular endothelial inflammation can be efficiently relieved. Inspired by this, we developed a miR-126-loaded immunoliposome with VCAM-1 monoclonal antibody (VCAMab) decorated at its surface. This immunoliposome can be directly targeted to VCAM-1 at the inflammatory vascular endothelial membrane surface and achieve highly efficient treatment against inflammation response. The cellular experiment results showed the immunoliposome had a higher uptake rate towards inflammatory human vein endothelial cells (HUVECs) and can significantly downregulate the VCAM-1 expression level of inflammatory HUVECs. In vivo investigation further demonstrated that this immunoliposome displayed a higher accumulation rate at vascular inflammatory dysfunction sites than its non-VCAMab-modified counterpart. These results suggest that this novel nanoplatform can effectively deliver miR-126 to vascular inflammatory endothelium, opening a new avenue for the safe and effective delivery of miRNA for potential clinical application.

The heparan sulfate mimetic Muparfostat aggravates steatohepatitis in obese mice due to its binding affinity to lipoprotein lipase.

Heparanase is the only confirmed endoglycosidase that cleaves heparan sulfate (HS), a ubiquitous glycosaminoglycan with various essential roles in multiple pathological processes. Thus, the development of heparanase inhibitors has become an attractive strategy for drug discovery, especially in tumour therapy, in which HS mimetics are the most promising compounds. The various biological effects of heparanase also suggest a role for HS mimetics in many non-cancer indications, such as type 1 diabetes. However, the potential benefits of HS mimetics in obesity-related type 2 diabetes have not been elucidated. In this study, we investigated muparfostat (PI-88), a developed HS mimetic currently enrolled in Phase III clinical trials, in obese mouse models and in vitro cultured murine hepatocytes. Daily administration of muparfostat for 4 weeks caused hyperlipidaemia and aggravated hepatic steatosis in obese mice models, but not in lean animals. In cultured hepatocytes, muparfostat did not alter lipid accumulation. Acute tests suggested that muparfostat binds to lipoprotein lipase in competition with HS on vascular endothelial cell surfaces, thereby reducing the degradation of circulating triglycerides by lipoprotein lipase and subsequent uptake of fatty acids into vascular endothelial cells and causing hyperlipidaemia. This hyperlipidaemia aggravates hepatic steatosis and causes liver injury in muparfostat-treated obese mice. The binding activity of HS mimetics to lipoprotein lipase should be investigated as an additional pharmacological effect during heparanase inhibitor drug discovery. This study also provides novel evidence for an increased risk of drug-induced liver injury in obese individuals.

Membrane Bending Enhances Sulfatide Mediated Sickle Mature Erythrocyte Adhesion

Introduction: Abnormal red blood cell (RBC) adhesion due to polymerization of sickle hemoglobin (HbS) is central to the pathophysiology of SCD. Early erythrocytes (reticulocytes), expanded in number due to sustained stress erythropoiesis stimulated by anemia and hypoxia, are more adhesive to vascular endothelial proteins and surfaces than are mature erythrocytes. However, mature erythrocytes typically constitute >80% of all RBCs in SCD, and the relative contribution made by erythrocytes vs. reticulocytes to acute and chronic vasculopathy is not well understood. We hypothesized that sphingomyelinase (SMase) activity that is upregulated by accumulated bending energy leads to elevated membrane sulfatide availability and thus hypoxic mature erythrocyte adhesion under hypoxia. Methods: We probed the adhesion of RBCs obtained from SCD patients (#NCT02824471) stained with acridine orange under various physiological conditions in microchannels that were coated with laminin. Then we analyzed the morphology of single sickle reticulocytes and mature erythrocytes to estimate the membrane bending energy. Results and Discussion: We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856±481 vs. 78±23, mean±SEM), while sickle reticulocyte adhesion, high to begin with, does not change (1281 ±299 vs. 1258±328, mean±SEM). Furthermore, we show that among the adherent RBCs, mature erythrocytes have greater elongation and accumulated membrane bending energy compared to reticulocytes under hypoxia (Figure 1A). Accumulated membrane bending is known to elicit SMase, and laminin establishes highly specific bonds with sulfatides which are downstream of the SMase. These results suggest that SMase is likely elicited particularly in sickle mature erythrocytes, which become highly adhesive with deoxygenation. This is likely due to loss of membrane surface area with RBC maturation, which may be excessive in SCD due to membrane vesiculation and microparticle release. To elucidate the roles of SMase and sulfatide in the adhesion of mature erythrocytes, we repeated the adhesion experiments with SMase and anti-sulfatide incubation. We found that SMase increases the hypoxia-enhanced adhesion of mature erythrocytes and the anti-sulfatide antibody inhibits the enhanced adhesion of mature erythrocytes seen under hypoxia (Figure 1B). Collectively, these data demonstrate the effect of accumulated membrane damage on the abnormal adhesion of sickle mature erythrocytes. Conclusions: Patient-to-patient variability in hypoxia enhanced mature erythrocyte adhesion suggests a role in modulating clinical comorbidities and or genetic polymorphisms relevant to red cell health and hypoxic regulation. These results also implicate the lipid components of the plasma membrane in the pathophysiology of RBC dysfunction in SCD. Therefore, sulfatide and the components of its pathway, such as SMase, could be explored as potential therapeutic targets to inhibit sickle erythrocyte adhesion in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Influence of α2-Macroglobulin, Anti-Parasite IgM and ABO Blood Group on Rosetting in Plasmodium falciparum Clinical Isolates and Their Associations with Disease Severity in a Ghanaian Population.

PurposeThe severity of Plasmodium falciparum infections is associated with the ability of the infected red blood cells to cytoadhere to host vascular endothelial surfaces and to uninfected RBCs. Host blood group antigens and two serum proteins α2-macroglobulin (α2M) and IgM have been implicated in rosette formation in laboratory-adapted P. falciparum. However, there is only limited information about these phenotypes in clinical isolates.MethodsThis was a hospital-based study involving children under 12 years-of-age reporting to the Hohoe Municipal Hospital with different clinical presentations of malaria. Parasite isolates were grown and rosette capabilities and characteristics were investigated by fluorescence microscopy. α2M and IgM were detected by ELISA.ResultsRosette formation was observed in 46.8% (75/160) of the parasite isolates from all the blood groups tested. Rosettes were more prevalent (55%) among isolates from patients with severe malaria compared to isolates from patients with uncomplicated malaria (45%). Rosette prevalence was highest (30%) among patients with blood group O (30%) and B (29%), while the mean rosette frequency was higher in isolates from patients with blood group A (28.7). Rosette formation correlated negatively with age (r = −0.09, P= 0.008). Participants with severe malaria had a lower IgM concentration (3.683±3.553) than those with uncomplicated malaria (5.256±4.294) and the difference was significant (P= 0.0228). The mean concentrations of anti-parasite IgM measured among the clinical isolates which formed rosettes was lower (4.2 ±3.930 mg/mL), than that in the non rosetting clinical isolates (4.604 ±4.159 mg/mL) but the difference was not significant (P=0.2733). There was no significant difference in plasma α2M concentration between rosetting and non rosetting isolates (P=0.442).ConclusionP. falciparum parasite rosette formation was affected by blood group type and plasma concentration of IgM. A lower IgM concentration was associated with severe malaria whilst a higher α2M concentration was associated with uncomplicated malaria.

Периферійна нейропатія при хламідійному реактивному артриті

Актуальність: периферійна нейропатія (ПНП) при реактивному хламідійному урогенітальному артриті (РХА) описується у вигляді поодиноких спостережень, і багато клініко-патогенетичних аспектів такого ураження нервової системи залишаються нез’ясованими. Мета дослідження: оцінити частоту й характер перебігу ПНП при РХА, взаємозв’язок нервових та суглобових ушкоджень, вивчити питання патогенетичних побудов цієї нейропатії, виділити чинники ризику. Матеріал і методи. Під наглядом перебував 101 хворий на РХА, середній вік яких становив 32 роки, тривалість захворювання — 4 роки, а співвідношення чоловіків і жінок — 1 : 1. У 90 % спостережень РХА виявлено Chlamydia trochamatis у секреті передміхурової залози, зіскобах з уретри, шийки матки, стінок піхви, у 83 % — позитивні серологічні тести на хламідійну інфекцію. Результати. При РХА ознаки ПНП мають місце в 19 % хворих у співвідношенні мононейропатія/полінейропатія як 1 : 1, із моторними, сенсорними і змішаними розладами в пропорції 1 : 3 : 6, наявністю вегетативних змін у кожного другого пацієнта та частішою дистальною локалізацією процесу в руках, на що впливають тяжкість перебігу суглобового синдрому, високі рівні протихламідійних антитіл у крові, а на аксональні й демієлінізуючі показники електронейроміографії — вираженість уражень урогеніталій і наявність синдрому Гійена — Барре. Високий показник темпів прогресування артриту є прогнознегативною ознакою перебігу ПНП у хворих на РХА. У патогенетичних побудовах ПНП беруть участь запальні імунні білки, порушення ендотеліальної функції судин та фізико-хімічні поверхневі реологічні властивості сироватки. Висновок. ПНП має місце в кожного п’ятого пацієнта з РХА, взаємопов’язана з клініко-лабораторними ознаками уражень суглобів, а в майбутньому буде корисним активне виявлення такої патології нервової системи для подальших своєчасних реабілітаційних заходів, причому серопозитивність РХА за антитілами до циклічного цитрулінового пептиду є чинником ризику такої ПНП.

Anti-phosphatidylserine/prothrombin complex antibodies in patients with cutaneous vasculitis: Possible involvement in the pathogenesis.

We assessed the IgG and IgM prevalence of anti-phosphatidylserine/prothrombin complex (aPS/PT) antibodies (Abs) in patients with vasculitis using a novel commercial ELISA kit. To examine whether aPS/PT Abs were involved in the pathogenesis of cutaneous vasculitis, inbred wild-type rats were intravenously administered with a rat IgM class aPS/PT monoclonal Ab established previously or with rat immunoglobulins as controls. To express PS on the surface of vascular endothelium, these rats were given a subcutaneous injection of cell-free histones in advance. Serum IgM aPS/PT Ab levels were elevated in patients with systemic vasculitis with skin involvement and cutaneous arteritis compared to those in patients with systemic vasculitis without skin involvement and healthy controls. There was no significant difference in the serum levels of IgG aPS/PT Abs between the patients and healthy controls. Correspondingly, inbred wild-type rats intravenously administered with the aPS/PT monoclonal IgM Ab after appropriate priming-subcutaneous histone injection developed cutaneous vasculitis. Some rats given rat IgM instead of the aPS/PT monoclonal Ab also developed cutaneous vasculitis, whereas vasculitis did not occur in rats given IgG or only priming by histones. We suggested that IgM aPS/PT Abs could be involved in the pathogenesis of cutaneous vasculitis based on these findings.

Three-dimensional electron microscopy for endothelial glycocalyx observation using Alcian blue with silver enhancement

Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies.

A novel biofunctionalizing peptide for metallic alloy.

Improving biocompatibility of metallic alloy biomaterials has been of great interest to prevent implant associated-diseases, such as stent thrombosis. Herein a simple and efficient procedure was designed to biofunctionalize a biomaterial surface by isolating a SUS316L stainless steel binding peptide. After three rounds of phage panning procedure, 12 mer peptide (SBP-A; VQHNTKYSVVIR) was identified as SUS316L-binding peptide. The SBP-A peptide formed a stable bond to a SUS316L modified surface and was not toxic to HUVECs. The SBP-A was then used for anti-ICAM antibody modification on SUS316L to construct a vascular endothelial cell-selective surface. The constructed surface dominantly immobilized vascular endothelial cells to smooth muscle cells, demonstrating that the SBP-A enabled simple immobilization of biomolecules without disturbing their active biological function. The SUS316L surface was successfully biofunctionalized using the novel isolated peptide SBP-A, showing its potential as an ideal interface molecule for stent modification. This is the first report of material binding peptide-based optimal surface functionalization to promote endothelialisation. This simple and efficient biofunctionalization procedure is expected to contribute to the development of biocompatible materials.

Contrast enhanced ultrasound molecular imaging of platelets in the inflammatory procession of atherosclerosis

Objective To assess the role of activated platelets in the inflammatory procession of atherosclerosis(AS) by ultrasound molecular imaging. Methods Sixty ApoE-/- mice were fed with high fat diet to establish AS model as experimental group, and 40 C57BL/6J mice were fed with normal diet as control group. Biotin-avidin bridging method was used to construct platelet-targeted microbubbles with recombinant vWF-A1 domain (Mb-A1), microbubbles carrying monoclonal antibodies to VCAM-1 (Mb-VCAM1) and microbubbles carrying IgG monoclonal antibody (Mb-ctrl). In vitro and in vivo experiments were carried out to evaluate the ability of Mb-A1 to target platelets on vascular endothelial surface. Contrast enhanced ultrasound molecular imaging of proximal ascending aorta was performed with Mb-A1, Mb-VCAM1 and Mb-ctrl. The expression and distribution of platelets and monocytes/macrophages on the endothelium of ascending aorta of AS mice were observed and analyzed by immunofluorescence staining. Results ①A large number of Mb-A1 adhering to the surface of activated platelets coated in Petri dishes were observed under fluoresce. ②After platelet immune-depletion in 30-week AS mice, the signal intensity of Mb-A1 decreased significantly in ascending aorta, while that of Mb-ctrl has no obvious change(P<0.05). ③In ApoE-/- mice, signals from platelet targeted microbubbles increased from 8 to 32 weeks of age in ApoE-/- mice, which coincided with the increase of signals from VCAM-1 targeted microbubbles(P<0.05). ④Activated platelets on the endothelial surface of ascending aorta increased progressively with age from 8 weeks, and partly overlapped with the distribution of monocytes/macrophages. Conclusions Platelets contribute to the initiation and progression of atherosclerosis as an inflammatory mediator through the interaction with vascular endothelium. Key words: Contrast enhanced ultrasound; Targeted microbubble; Atherosclerosis; Inflammation; Platelet

Endothelial glycocalyx as a potential theriapeutic target in organ injuries.

Objective:The endothelial glycocalyx (eGC) is a dynamic and multicomponent layer of macromolecules found at the surface of vascular endothelium, which is largely underappreciated. It has recently been recognized that eGC is a major regulator of endothelial function and may have therapeutic value in organ injuries. This study aimed to explore the role of the eGC in various pathologic and physiologic conditions, by reviewing the basic research findings pertaining to the detection of the eGC and its clinical significance. We also explored different pharmacologic agents used to protect and rebuild the eGC.Data sources:An in-depth search was performed in the PubMed database, focusing on research published after 2003 with keywords including eGC, permeability, glycocalyx and injuries, and glycocalyx protection.Study selection:Several authoritative reviews and original studies were identified and reviewed to summarize the characteristics of the eGC under physiologic and pathologic conditions as well as the detection and protection of the eGC.Results:The eGC degradation is closely associated with pathophysiologic changes such as vascular permeability, edema formation, mechanotransduction, and clotting cascade, together with neutrophil and platelet adhesion in diverse injury and disease states including inflammation (sepsis and trauma), ischemia-reperfusion injury, shock, hypervolemia, hypertension, hyperglycemia, and high Na+ as well as diabetes and atherosclerosis. Therapeutic strategies for protecting and rebuilding the eGC should be explored through experimental test and clinical verifications.Conclusions:Disturbance of the eGC usually occurs at early stages of various clinical pathophysiologies which can be partly prevented and reversed by protecting and restoring the eGC. The eGC seems to be a promising diagnostic biomarker and therapeutic target in clinical settings.

Factor H interferes with the adhesion of sickle red cells to vascular endothelium: a novel disease-modulating molecule.

Sickle cell disease is an autosomal recessive genetic red cell disorder with a worldwide distribution. Growing evidence suggests a possible involvement of complement activation in the severity of clinical complications of sickle cell disease. In this study we found activation of the alternative complement pathway with microvascular deposition of C5b-9 on skin biopsies from patients with sickle cell disease. There was also deposition of C3b on sickle red cell membranes, which is promoted locally by the exposure of phosphatidylserine. In addition, we showed for the first time a peculiar “stop-and-go” motion of sickle cell red blood cells on tumor factor-α–activated vascular endothelial surfaces. Using the C3b/iC3b binding plasma protein factor Has an inhibitor of C3b cell-cell interactions, we found that factor H and its domains 19-20 prevent the adhesion of sickle red cells to the endothelium, normalizing speed transition times of red cells. We documented that factor H acts by preventing the adhesion of sickle red cells to P-selectin and/or the Mac-1 receptor (CD11b/CD18), supporting the activation of the alternative pathway of complement as an additional mechanism in the pathogenesis of acute sickle cell related vaso-occlusive crises. Our data provide a rationale for further investigation of the potential contribution of factor H and other modulators of the alternative complement pathway with potential implications for the treatment of sickle cell disease.

271 Imaging the placental glycocalyx

Introduction Glycocalyx is a gel-like mesh which lines the luminal surface of vascular endothelium and has an important role in vessel function and permeability. Disruption of glycocalyx has been implicated in the endothelial dysfunction in pre-eclampsia. There is increasing evidence for a significant glycocalyx present at the maternal-fetal interface of the human placenta. Due to the unstable nature of glycocalyx ex-vivo it has previously been difficult to demonstrate and study. Objectives To confirm the presence of the placental glycocalyx of the fetal capillary endothelium and syncytiotrophoblast and to develop a reliable imaging protocol to observe this ex-vivo. Methods Term placentae were collected from uncomplicated pregnancies at caesarean section and processed in one of two ways. 1) Electron Microscopy: placental glycocalyx was labelled using perfusion or immersion with Alcian Blue in glutaraldehyde. Samples were post fixed, embedded and sectioned for imaging with transmission electron microscopy (TEM). Glycocalyx depth was measured perpendicular to the phospholipid bilayer. 2) Lectin Staining. Biopsies were fixed in 4% paraformaldehyde, paraffin embedded and sectioned. Glycocalyx was labelled with FITC-conjugated wheat germ agglutinin (WGA) and membrane labelled with octadecyl rhodamine B chloride (R18) and imaged using confocal microscopy. The distance between the peak signals of FITC-WGA and R18 was used to measure glycocalyx depth. Results presented as mean glycocalyx thickness ±SEM. Results TEM demonstrated glycocalyx depth of 57.1 ± 5.8 nm at the fetal capillary and 62.2 ± 3.7 nm at the syncytiotrophoblast. Glycocalyx depth using lectin staining was significantly greater 279.3 ± 42.6 nm and 893.7 ± 150.1 nm at the capillary and syncytiotrophoblast respectively. Conclusion The placenta maintains a significant glycocalyx at the syncytiotrophoblast and fetal capillary endothelium in term uncomplicated pregnancy. The absolute depth of glycocalyx varied depending on the imaging modality used. Correlative fluorescence and electron microscopy techniques would help further define this.

Predictors for the efficacy of Endostar combined with neoadjuvant chemotherapy for stage IIIA (N2) NSCLC.

Endostar (rh-endostatin) is a new recombinant human endostatin, which could inhibit cell proliferation, angiogenesis, and tumor growth. To explore anti-angiogenesis short-term efficacy combined with neoadjuvant chemotherapy for stage IIIA (N2) non-small cell lung cancer (NSCLC), and identify the potential predictive factors. We pathologically examined 26 patients diagnosed with stage IIIA (N2) NSCLC who received NP chemotherapy alone or combined with Endostar, respectively. Our results indicated that total clinical benefit rate (CBR) 87.5% and 64% (p= 0.76), respectively. The clinical benefit (CB) patients in the treatment group showed significant changes in endothelial progenitor cells (EPC), vascular endothelial growth factor (VEGF), blood flow (BF), permeability surface (PMS), and microvascular density (MVD) before and after treatment. Compared with CB patients in the control group, changes in EPC and MVD (only) before and after treatment were significant. The variation of EPC, PMS, and MVD before and after treatment in the treatment group showed positive correlation with tumor regression rate (TRR) and the variation of MVD, whereas those of EPC and PMS demonstrated positive correlations with variation of MVD before and after treatment. Our findings suggested that PMS and EPC may be used as a predictive factor for the short-term efficacy of the combined therapy in NSCLC.

Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression

L-selectin (CD62L) is an extracellular protein with a lectin-like domain that mediates rolling adhesion of leukocytes to vascular endothelial cell surfaces. Currently, there are no solved structures for the ectodomain of CD62L, nor of CD62L in complex with its ligand. We have developed a rapid mammalian recombinant protein expression system using an amplifiable glutamine synthase based vector. Here, we further developed and applied this method to express and purify the entire extracellular region of CD62L. This resulted in excess of 20 mg/L yield of recombinant CD62L. In an attempt to understand the different expression levels among four similar CD62L constructs that differ primarily in signal sequences, we calculated the presence of potential RNA pseudoknots in their signal sequences. The results showed the presence of pseudoknots involving the start codon and between the signal sequence and gene in the mRNA of the non-expressing constructs, suggesting a potential inhibitory role of RNA pseudoknots in recombinant protein expression.

Biosynthesis and Functional Significance of Peripheral Node Addressin in Cancer-Associated TLO

Peripheral node addressin (PNAd) marks high endothelial venules (HEV), which are crucial for the recruitment of lymphocytes into lymphoid organs in non-mucosal tissue sites. PNAd is a sulfated and fucosylated glycoprotein recognized by the prototypic monoclonal antibody, MECA-79. PNAd is the ligand for L-selectin, which is expressed on the surface of naive and central memory T cells, where it mediates leukocyte rolling on vascular endothelial surfaces. Although PNAd was first identified in the HEV of peripheral lymph nodes, recent work suggests a critical role for PNAd in the context of chronic inflammatory diseases, where it can be used as a marker for the formation of tertiary lymphoid organs (TLOs). TLO form in tissues impacted by sustained inflammation, such as the tumor microenvironment where they function as local sites of adaptive immune cell priming. This allows for specific B- and T-cell responses to be initiated or reactivated in inflamed tissues without dependency on secondary lymphoid organs. Recent studies of cancer in mice and humans have identified PNAd as a biomarker of improved disease prognosis. Blockade of PNAd or its ligand, L-selectin, can abrogate protective antitumor immunity in murine models. This review examines pathways regulating PNAd biosynthesis by the endothelial cells integral to HEV and the formation and maintenance of lymphoid structures throughout the body, particularly in the setting of cancer.

Impact of Blood Groups on Clinical Outcomes in Patients with Von Willebrand Disease

INTRODUCTIONVon Willebrand Disease (vWD) is the most common hereditary hemorrhagic disorder with diverse clinical and genetic characteristics. Transmitted as an autosomal dominant trait (except Type III), it is caused primarily by the quantitative or qualitative deficiency of von Willebrand factor (vWF). ABO blood group types can affect the pathology, severity and hence the presentation of this clinically heterogeneous entity as the genes responsible for ABO blood groups also affects vWF levels in the plasma. We conducted a study to analyze and compare different clinico-demographic aspects among O and non-O blood group patients with Von Willebrand Disease. Our primary aim was to determine the relationship of different blood groups with venous thromboembolism (VTE) in patient with vWD. Our secondary aim was to determine the association of blood groups with different comorbid conditions such as coronary artery disease, cerebrovascular disease, peripheral vascular disease, chronic liver disease, chronic kidney disease etc. in patients with vWD.METHODSA retrospective review was carried out to include all patients diagnosed with Von Willebrand Disease from year 2002 to 2012. Patient population was obtained by using International Classification of Diseases, 9th revision (ICD9) codes. Data was collected with the help of electronic medical records. Multiple clinical variables and demographic characteristics of Von Willebrand Disease patients (n=381) were analyzed and compared between those with type O blood group (205 patients; 54%) and those with type non-O blood groups (176 patients; 46%). Univariate logistic regression was used to obtain crude odds ratios. Predictors with statistically significant p-values were included in a multivariable logistic regression model in order to yield adjusted odds ratio. Statistical significance was set at p<0.05RESULTSPatients with O blood groups were less likely to develop VTE (OR 0.29, 95% CI 0.16-0.52, p < 0.001) as compared to Non-O blood groups in which the prevalence of VTE was much higher (25% vs. 9%). Additionally, patients with O blood groups had relatively lower factor 8 activity (125.9±90.8%; OR 0.99, 95% CI 0.99-1.00, p = 0.018) when compared with Non-O blood groups (168.5±120.6%). Patients with O-blood group had significant, life threatening bleeds at an early age (40.4±20.8 years; OR 0.99, 95% CI 0.97-1.00, p = 0.008) when compared with patients with type non-O blood group (Age 47.1±21.9 years). Additionally, O-Blood groups were related to a lesser prevalence of cerebrovascular disease (OR 0.48, 95% CI 0.29-0.79, p = 0.004) and peripheral vascular disease (OR 0.47, 95% CI 0.25-0.87, p = 0.015) as compared to Non-O blood groups.DISCUSSIONOur study consolidates the fact that vWD patients with O blood group have a lesser risk of developing VTE as compared to non-O blood group patients. Additionally, strokes and peripheral vascular disease is also seen less frequently in O blood groups in patients with Von Willebrand disease. These results suggest that the genes responsible for ABO blood groups also have a significant effect on clinical outcomes in patients with vWD. There are multiple proposed mechanisms to explain this association including presence of ABO antigens on several platelet glycoproteins and glycosphingolipids as well as on the surface of vascular endothelium. More sophisticated, large-scale studies are needed to strengthen the associations we observed in our retrospective study. DisclosuresKuriakose:Kedrion: Speakers Bureau.

Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats.

BackgroundThe beverage obtained by fermentation of milk with kefir grains, a complex matrix containing acid bacteria and yeasts, has been shown to have beneficial effects in various diseases. However, its effects on hypertension and endothelial dysfunction are not yet clear. In this study, we evaluated the effects of kefir on endothelial cells and vascular responsiveness in spontaneously hypertensive rats (SHR).MethodsSHR were treated with kefir (0.3 mL/100 g body weight) for 7, 15, 30 and 60 days and compared with non-treated SHR and with normotensive Wistar-Kyoto rats. Vascular endothelial function was evaluated in aortic rings through the relaxation response to acetylcholine (ACh). The balance between reactive oxygen species (ROS) and nitric oxide (NO) synthase was evaluated through specific blockers in the ACh-induced responses and through flow cytometry in vascular tissue.ResultsSignificant effects of kefir were observed only after treatment for 60 days. The high blood pressure and tachycardia exhibited by the SHR were attenuated by approximately 15 % in the SHR-kefir group. The impaired ACh-induced relaxation of the aortic rings observed in the SHR (37 ± 4 %, compared to the Wistar rats: 74 ± 5 %), was significantly attenuated in the SHR group chronically treated with kefir (52 ± 4 %). The difference in the area under the curve between before and after the NADPH oxidase blockade or NO synthase blockade of aortic rings from SHR were of approximately +90 and −60 %, respectively, when compared with Wistar rats. In the aortic rings from the SHR-kefir group, these values were reduced to +50 and −40 %, respectively. Flow cytometric analysis of aortic endothelial cells revealed increased ROS production and decreased NO bioavailability in the SHR, which were significantly attenuated by the treatment with kefir. Scanning electronic microscopy showed vascular endothelial surface injury in SHR, which was partially protected following administration of kefir for 60 days. In addition, the recruitment of endothelial progenitor cells was decreased in the non-treated SHR and partially restored by kefir treatment.ConclusionsKefir treatment for 60 days was able to improve the endothelial function in SHR by partially restoring the ROS/NO imbalance and the endothelial architecture due to endothelial progenitor cells recruitment.

N-glycans and metastasis in galectin-3 transgenic mice

Poly-N-acetyl-lactosamine (polyLacNAc) on N-glycans facilitate lung specific metastasis of melanoma cells by serving as high affinity ligands for galectin-3, expressed in highest amounts in the lungs, on almost all its tissue compartments including on the surface of vascular endothelium. PolyLacNAc not only aids in initial arrest on the organ endothelium but in all the events of extravasation. Inhibition of polyLacNAc synthesis, or competitive inhibition of its interaction with galectin-3 all inhibited these processes and experimental metastasis. Transgenic galectin-3 mice, viz., gal-3+/+ (wild type), gal-3+/− (hemizygous) and gal-3−/− (null) have been used to prove that galectin-3/polyLacNAc interactions are indeed critical for lung specific metastasis.Gal-3+/− mice which showed <50% expression of galectin-3 on the lungs also showed proportionate decrease in the number of B16F10 melanoma metastatic colonies affirming that galectin-3 and polyLacNAc interactions are indeed key determinants of lung metastasis. However, surprisingly, the number and size of metastatic colonies in gal-3−/− mice was very similar as that seen in gal-3+/+ mice. The levels of lactose binding lectins on the lungs and the transcripts of other galectins (galectin-1, -8 and -9) which are expressed on lungs and have similar sugar binding specificities as galectins-3, remain unchanged in gal-3+/+ and gal-3−/− mice. Further, inhibition of N-glycosylation with Swainsonine (SW) which drastically reduces metastasis of B16F10 cells in gal-3+/+ mice, did not affect lung metastasis when assessed in gal-3−/− mice. Together, these results rule out the possibility of some other galectin taking over the function of galectin-3 in gal-3−/− mice. Chimeric mice generated to assess if absence of any effect on metastasis is due to compromised tumor immunity by replacing bone marrow of gal-3−/− mice with that from gal-3+/+ mice, also failed to impact melanoma metastasis. As galectin-3 regulates several immune functions including maturation of different immune cells, compromised tumor immunity could be the major determinant of melanoma metastasis in gal-3−/− mice and warrants thorough investigation.