Endothelial Survival Research Articles

Shear Stress Regulates Endothelial Microparticle Release

Endothelial activation and apoptosis release membrane-shed microparticles (EMP) that emerge as important biological effectors. Because laminar shear stress (SS) is a major physiological regulator of endothelial survival, we tested the hypothesis that SS regulates EMP release. EMP levels were quantified by flow cytometry in medium of endothelial cells subjected to low or high SS (2 and 20 dyne/cm(2)). EMP levels augmented with time in low SS conditions compared with high SS conditions. This effect was sensitive to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and Rho kinases inhibitors but unaffected by caspase inhibitors. Low SS-stimulated EMP release was associated with increased endothelial Rho kinases and ERK1/2 activities and cytoskeletal reorganization. Overexpression of constitutively active RhoA stimulated EMP release under high SS. We also examined the effect of nitric oxide (NO) in mediating SS effects. L-NG-nitroarginine methyl ester (L-NAME), but not D-NG-nitroarginine methyl ester, increased high SS-induced EMP levels by 3-fold, whereas the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) decreased it. L-NAME and SNAP did not affect Rho kinases and ERK1/2 activities. Then, we investigated NO effect on membrane remodeling because microparticle release is abolished in ABCA1-deficient cells. ABCA1 expression, which was greater under low SS than under high SS, was augmented by L-NAME under high SS and decreased by SNAP under low SS conditions. Altogether, these results demonstrate that sustained atheroprone low SS stimulates EMP release through activation of Rho kinases and ERK1/2 pathways, whereas atheroprotective high SS limits EMP release in a NO-dependent regulation of ABCA1 expression and of cytoskeletal reorganization. These findings, therefore, identify endothelial SS as a physiological regulator of microparticle release.

Endothelial Keratoplasty: Prospective, Randomized, Masked Clinical Trial Comparing an Injector With Forceps for Tissue Insertion

To compare the complications and outcomes of Descemet stripping automated endothelial keratoplasty (DSAEK) when the tissue is either folded and inserted with a forceps or inserted using a platform injector device without folding. Prospective, randomized, masked clinical trial. DSAEK was performed in 100 eyes of 79patients undergoing DSAEK surgery for Fuchs corneal dystrophy. Fifty eyes were randomized to have the donor tissue inserted with Charlie II insertion forceps (Bausch & Lomb Surgical) and 50 eyes were randomized to have the donor tissue inserted with the Neusidl Corneal Inserter (Fischer Surgical Inc). All other steps of the surgical procedure were exactly the same. Surgical problems, postoperative complications, and central endothelial cell density at 6months were recorded and then measured by a masked observer. The study's main outcome measures were total central endothelial cell density and percentage of donor endothelial cell loss from before surgery to 6months after surgery and rate of complications (graft dislocation and primary graft failure). No primary graft failures occurred in either group and only 1 dislocation occurred in the series (Neusidl group). One late failure occurred at 6months (Neusidl group). There was no difference in the preoperative endothelial cell density between the Neusidl and forceps groups, but there was a higher percentage of cell loss with the Neusidl group (33%) than with the forceps group (25%) at 6months (P= .017). The Neusidl Corneal Inserter yielded a low immediate complication rate for DSAEK surgery for novice and experienced surgeons. Although still at an acceptable level, short-term endothelial survival was significantly worse after Neusidl tissue insertion than that after forceps tissue insertion.

Human chorionic gonadotropin controls luteal vascular permeability via vascular endothelial growth factor by down-regulation of a cascade of adhesion proteins

To study the functional interactions of junctional proteins acting as regulators of vascular permeability in the human corpus luteum. We investigated the role of vascular endothelial (VE)-cadherin, nectin 2, and claudin 5 as controllers of vascular endothelial cell permeability. Performing immunohistochemical dual staining, we colocalized the above-mentioned proteins in the human corpus luteum. Not applicable. Not applicable. Not applicable. Using a granulosa-endothelial coculture system, we revealed that hCG-treatment down-regulates VE-cadherin, nectin 2, and claudin 5 in endothelial cells via vascular endothelial growth factor (VEGFA). Furthermore, the interaction of VE-cadherin, nectin 2, and claudin 5 was investigated by silencing these proteins that perform siRNA knockdown. Interestingly, knockdown of VE-cadherin and claudin 5 induced a decrease of the respective other protein. This down-regulation was associated with changed rates of vascular permeability: hCG induced a VEGFA-dependent down-regulation of VE-cadherin, nectin 2, and claudin 5, which increased the endothelial permeability in the coculture system. Furthermore, knockdown of VE-cadherin, nectin-2, and claudin 5 also resulted in a consecutive increase of endothelial permeability for each different protein. These results demonstrate for the first time that VE-cadherin, nectin 2, and claudin 5 are involved in the regulation of vascular permeability in a mutually interacting manner, which indicates their prominent role for the functionality of the human corpus luteum.

Circulating Angiopoietin-2 Is a Marker for Early Cardiovascular Disease in Children on Chronic Dialysis

Cardiovascular disease (CVD) is increasingly recognised as a complication of childhood chronic kidney disease (CKD) even in the absence of diabetes and hypertension. We hypothesized that an alteration in angiopoietin-1 and -2, growth factors which regulate endothelial and vascular function could be involved. We report that the endothelial survival factor, angiopoietin-1 is low in children with pre-dialysis CKD whereas the pro-inflammatory angiopoietin-2 is elevated in children on dialysis. In dialysis patients, angiopoietin-2 positively correlated with time on dialysis, systolic blood pressure, and carotid artery intima media thickness. Elevated angiopoietin-2 levels in dialysis versus pre-dialysis CKD patients were also associated with an anti-angiogenic (high soluble VEGFR-1 and low VEGF-A) and pro-inflammatory (high urate, E-selectin, P-selectin and VCAM-1) milieu. Ang-2 was immunodetected in arterial biopsy samples whilst the expression of VEGF-A was significantly downregulated in dialysis patients. Serum urate correlated with angiopoietin-2 levels in dialysis patients and addition of uric acid was able to induce rapid release of angiopoietin-2 from cultured endothelial cells. Thus, angiopoietin-2 is a marker for cardiovascular disease in children on chronic dialysis and may act as an anti-angiogenic and pro-inflammatory effector in this context. The possibility that the release of angiopoietin-2 from endothelia is mediated by urate should be explored further.

High phosphate directly affects endothelial function by downregulating annexin II

Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.

The angiogenic factors and their soluble receptors in sepsis: friend, foe, or both?

The endothelium represents an important source of, and a target for, inflammation in sepsis. Angiogenic factors and their receptors, including vascular endothelial growth factor (VEGF)/VEGF receptor and the angiopoietin/Tie2 signaling pathways, have recently received great attention in critically ill patients, including those with sepsis [1-3], because of their pivotal roles in both angiogenesis and microvascular permeability. VEGF, as the most potent proangiogenic factor, has already been identified as an important target for cancer therapy. Disassembly of an intact endothelial cell junction is necessary for the angiogenesis process. The endothelial barrier-breaking properties are indeed an important part of the VEGF role in the regulation of angiogenesis. However, a high dose or prolonged duration of VEGF may lead to excessive barrier-breaking effects. The favorable results observed with the only US Food and Drug Administration-approved anti-VEGF agent, bevacizumab, by Jeong and colleagues [4] are likely to provide potential advances for future therapeutic opportunities in sepsis. On the contrary, VEGF shows endothelial survival signals, and suppression of VEGF signaling inhibits endothelial survival and increases apoptosis, which in turn may contribute to the development of sepsis-induced organ dysfunction [5]. Furthermore, other than an independent function on endothelial cells, VEGF plays an important role in mobilizing endothelial progenitor cells under pathologic conditions such as cancer and sepsis. While the decrease of circulating endothelial progenitor cells (cEPCs) after anti-VEGF treatment is beneficial in cancer, this may not be so in sepsis. The endothelium is a chief target of sepsis-induced events, and clinical outcome in septic patients is largely dependent on the ability to reconstitute damaged endothelium. Indeed, several studies have observed an increase in cEPC numbers in septic patients and a correlation between cEPC concentration and survival. The increased number of cEPCs in sepsis might therefore be a consequence of the body’s attempt to limit vascular damage by inducing endogenous endothelial repair mechanisms. The dramatic decrease of cEPCs accompanied by anti-VEGF treatment may impair endothelial repair capacity and bring unfavorable effects in sepsis. The angiogenic factors and their soluble receptors therefore do have a complex role and seemingly play both good and bad roles during sepsis development and therapy. A certain level of VEGF is necessary for normalizing endothelial function, and abnormally high or low levels of VEGF might shift its role from endothelial protective to endothelial barrier disruptive. This view can also provide additional possible explanations for why Jeong and colleagues’ study suggests that a high dose of bevacizumab may have deleterious effects in an experimental sepsis model. Future studies on the dynamic change of the angiogenic factors, their soluble receptors and cEPC counts during and after each therapy, as well as the context-dependent role of them, may provide background data for using these angiogenic factors and their soluble receptors as therapeutic targets for sepsis treatment in clinical practice.

Association between Pretreatment Levels of Serum Vascular Endothelial Growth Factor (VEGF) and Survival Outcomes in Locally Advanced Cervical Cancer Patients

Objective: To evaluate the association between pretreatment levels of serum vascular endothelial growth factor (VEGF) and long-term treatment outcomes in patients with locally advanced cervical cancer (LACC). Methods: Thirty-nine patients diagnosed with LACC (stage IIB-IVA) and obtaining blood for serum VEGF were identified. All patients received complete treatment as radical radiotherapy with or without concurrent chemotherapy. Surveillance for all patients was every 3 months during the first 2 years, and every 6 months later. Results: Mean age of 39 patients was 52.3 ± 10.8 years old. Twenty-three patients (59.0%) had stage IIB, and 16 patients (41.0%) had stage IIIB. Histological cell type was mostly squamous cell carcinoma (89.7%). The median and 75th percentile level of serum VEGF were 610.2 pg/ml (0.0 - 4067.2 pg/ml) and 825.6 pg/ml, respectively. At median follow-up of 37.0 months (range, 26.8 - 46.3 months), the 3-year OS rate was 78.6%. Clinical stage (p = 0.04) and 75th percentile of VEGF level (p = 0.04) were impacted on OS in univariable analysis. The 3-year OS of patients in stage IIB with serum VEGF of ≤825.6 pg/ml and of > 825.6 pg/ml was slightly different, 94.4% and 80.0% respectively (p = 0.34), whereas there were many differences in stage IIIB, 71.4% and 25.0% in patients with serum VEGF of ≤825.6 pg/ml and of >825.6 pg/ml respectively (p = 0.05). Conclusion: High pretreatment serum VEGF level has an influence on OS for LACC. It is potentially used as a predictive factor, especially in patients stage IIIB, in order to provide efficient treatment and improve survival outcomes in the future.

RhoB Differentially Controls Akt Function in Tumor Cells and Stromal Endothelial Cells during Breast Tumorigenesis

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

Abstract 18700: Accelerated Endothelial Regeneration and Survival Attenuate Vein Graft Atherosclerosis via Terahydrobiopterin-Dependent eNOS Coupling

As a result of surgical trauma, ischemia and exposure to arterial pressure the majority of endothelium in vein grafts (VG) undergo necrosis or apoptosis. We hypothesized that increased eNOS coupling could protect endothelial viability after VG surgery. To investigate this we utilized a transgenic mouse with endothelial-targeted overexpression of the essential eNOS coupling cofactor BH4. We investigated the effect of BH4 on endothelial cell survival and vascular remodeling in vitro and in vivo using ApoE-KO mice with endothelial-targeted overexpression of the rate limiting enzyme of BH4 synthesis GTP cyclohydrolase 1 (GCH) and LacZ reporter gene. BH4 levels were increased 10-fold in primary endothelium and 5-fold in aorta from GCH/ApoE-KO mice compared with ApoE-KO, without concomitant increases in eNOS protein. Despite no differences at baseline, following 4 days of culture, total endothelial cell number, colony size and number from GCH/ApoE-KO mice were increased (5-fold P=0.05; 2-fold P=0.02; 2-fold P=0.06 respectively, n=6) as were putative EPC levels from bone-marrow mononuclear cells determined by culture (15%,P=0.04) and FACS (26%, P=0.03). Vascular injury as a result of VG surgery in ApoE-KO mice led to significant neointima that was augmented by 61% (P=0.002) in GCH/ApoE-KO mice. Furthermore, endothelial repopulation was accelerated by 21% (P=0.01) and endothelial survival 44% (P=0.03) in these animals. O2- production was significantly attenuated in GCH/ApoE-KO mice measured on both aortic sections using oxidative microtopography and whole aorta by lucigenin chemiluminescence. eNOS inhibition by L-NAME reversed these effects indicating that under basal conditions GCH/ApoE-KO mice had preserved eNOS coupling promoting NO synthesis. In keeping with these findings in 219 patients with coronary artery disease, analysis of saphenous vein and internal mammary artery identified that high vascular BH4 levels were associated with greater vasorelaxations to acetylcholine (P<0.05) and reduced total L-NAME inhibitable O2- suggesting improved eNOS coupling. Availability of BH4 within the endothelium critically modulates the response to vascular injury by maintaining eNOS coupling and accelerating endothelial regeneration.

Transgenic Expression of FoxM1 Promotes Endothelial Repair following Lung Injury Induced by Polymicrobial Sepsis in Mice

Enhancing endothelial barrier integrity for the treatment of acute lung injury (ALI) is an emerging novel therapeutic strategy. Our previous studies have demonstrated the essential role of FoxM1 in mediating endothelial regeneration and barrier repair following lipopolysaccharide-induced lung injury. However, it remains unclear whether FoxM1 expression is sufficient to promote endothelial repair in experimental models of sepsis. Here, employing the FoxM1 transgenic (FoxM1 Tg) mice, we showed that transgenic expression of FoxM1 promoted rapid recovery of endothelial barrier function and survival in a clinically relevant model of sepsis induced by cecal ligation and puncture (CLP). We observed lung vascular permeability was rapidly recovered and returned to levels similar to baseline at 48 h post-CLP challenge in FoxM1 Tg mice whereas it remained markedly elevated in WT mice. Lung edema and inflammation were resolved only in FoxM1 Tg mice at 24 h post-CLP. 5-bromo-2-deoxyuridine incorporation assay revealed a drastic induction of endothelial proliferation in FoxM1 Tg lungs at 24h post-CLP, correlating with early induction of expression of FoxM1 target genes essential for cell cycle progression. Additionally, deletion of FoxM1 in endothelial cells, employing the mouse model with endothelial cell-restricted disruption of FoxM1 (FoxM1 CKO) resulted in impaired endothelial repair following CLP challenge. Together, these data suggest FoxM1 expression in endothelial cells is necessary and sufficient to mediate endothelial repair and thereby promote survival following sepsis challenge.

Heme Binding Proteins of Bartonella henselae Are Required when Undergoing Oxidative Stress During Cell and Flea Invasion

Bartonella are hemotropic bacteria responsible for emerging zoonoses. These heme auxotroph alphaproteobacteria must import heme for their growth, since they cannot synthesize it. To import exogenous heme, Bartonella genomes encode for a complete heme uptake system enabling transportation of this compound into the cytoplasm and degrading it to release iron. In addition, these bacteria encode for four or five outer membrane heme binding proteins (Hbps). The structural genes of these highly homologous proteins are expressed differently depending on oxygen, temperature and heme concentrations. These proteins were hypothesized as being involved in various cellular processes according to their ability to bind heme and their regulation profile. In this report, we investigated the roles of the four Hbps of Bartonella henselae, responsible for cat scratch disease. We show that Hbps can bind heme in vitro. They are able to enhance the efficiency of heme uptake when co-expressed with a heme transporter in Escherichia coli. Using B. henselae Hbp knockdown mutants, we show that these proteins are involved in defense against the oxidative stress, colonization of human endothelial cell and survival in the flea.

Comparison of Penetrating Keratoplasty and Deep Lamellar Keratoplasty for Macular Corneal Dystrophy and Risk Factors of Recurrence

To compare the therapeutic effects of penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK) on patients with macular corneal dystrophy (MCD) and to analyze the risk factors of postoperative recurrence. Retrospective, interventional, comparative case series. Fifty-one patients (78 eyes) with MCD treated by PK or DALK at Shandong Eye Institute between January 1992 and December 2010. The medical records of the patients were reviewed retrospectively. Best-corrected visual acuity, corneal endothelial density, complications, recurrence, graft survival, and risk factors for recurrence. Penetrating keratoplasty was performed in 57 eyes, and DALK was performed in 21 eyes. The mean follow-up time was 5.1±4.1 years (range, 1.0-18.0 years). The best-corrected visual acuity of the PK group was much better than that of the DALK group at 1, 2, 3, and 5 years. The corneal endothelial density was reduced to 1000 cells/mm2 or less within 5 years in 21.6% (11/51) of eyes treated by PK and in none of the eyes treated by DALK. The 1-year incidence rate of complications was 21.1% in the PK group, higher than the 4.8% rate in the DALK group. At the last visit, the rate of graft clarity was 87.7% and 85.7% in the 2 groups, respectively. Ten eyes (17.5%) treated by PK had recurrent MCD, with a rate of 0.8%, 7.7%, and 40% at 1, 5, and 10 years, respectively, whereas 9 eyes (42.9%) treated by DALK demonstrated recurrence, with a rate of 14.3% and 49.5% at 1 and 5 years, respectively. The recurrence risk was higher in patients whose age was 18 years or younger at onset or younger than 30 years at surgery. The recurrence risk after DALK was 5.066 times higher than that after PK. Penetrating keratoplasty more often immediately improves the visual acuity of patients with MCD, but many complications seem to be inevitable, especially continuous loss of corneal endothelium. Despite poor visual acuity and recurrence after surgery, DALK may produce fewer complications overall and more durable stability of the ocular surface compared with PK. The selection of PK or DALK for MCD should depend on the actual need and situation of certain patients. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Facio-Genital Dysplasia-5 Regulates Matrix Adhesion and Survival of Human Endothelial Cells

The function of the endothelial cell (EC)-enriched Rho family guanine nucleotide exchange factor, facio-genital dysplasia-5 (FGD5), is poorly understood. We sought to determine whether FGD5 regulates endothelial cytoskeletal reorganization and angiogenesis. We observed that FGD5 is expressed in primary human EC isolated from sites across the vasculature. Inhibition of FGD5 expression using RNA interference decreased the protein by ≈70%. In 3-dimensional vascular endothelial growth factor-stimulated angiogenesis in vitro, FGD5-deficient endothelial sprout protrusion was markedly blunted versus nonsilenced controls. FGD5 knockdown impaired adhesion to fibronectin and collagen IV and remodeling of matrix adhesion complexes. Similarly, monolayer electric impedance was decreased, and impedance increased at a slower rate after seeding FGD5-deficient cells versus controls, reflecting decreased EC spreading. Further, FGD5 plays a role in cell survival, because expression of cleaved caspase-3 was increased in FGD5-deficient EC after loss of cell-matrix contacts, and proapoptotic tumor necrosis factor-α stimulation elicited EC with subdiploid DNA content among FGD5-deficient EC. Mechanistically, the phosphatidylinositol 3-kinase/Akt pathway that regulates both adhesive and survival signal transduction pathways requires FGD5. Vascular endothelial growth factor-stimulated Akt phosphorylation and downstream forkhead box protein-O1 inactivation is inhibited by FGD5 loss. FGD5 regulates endothelial adhesion, survival, and angiogenesis by modulating phosphatidylinositol 3-kinase signaling.

Endothelial Cell Loss and Graft Survival after Descemet's Stripping Automated Endothelial Keratoplasty and Penetrating Keratoplasty

We sought to compare endothelial cell (EC) loss and graft survival after Descemet's stripping automated endothelial keratoplasty (DSAEK) and conventional penetrating keratoplasty (PK) in Asian eyes. Retrospective, nested, cohort study. Consecutive patients who underwent DSAEK or PK with Fuchs' endothelial dystrophy or pseudophakic and aphakic bullous keratopathy (BK) at a single tertiary center from April 1, 2006, to April 1, 2008. Clinical data, and donor and recipient characteristics were recorded from our prospective cohort from the Singapore Corneal Transplant Study. Percent EC loss and graft survival up to 3 years. There were no differences in baseline characteristics of patients comparing DSAEK and PK; there were more patients with BK (n = 141, 68.4%) than Fuchs' dystrophy. Percent EC loss was lower in DSAEK compared with PK at 1 (30 ± 22% vs 37 ± 25%; P = 0.045), 2 (36 ± 23% vs 45 ± 33%; P = 0.018), and 3 years (39 ± 24% vs 47 ± 28%; P = 0.022) postoperatively. Graft survival was comparable (log-rank P = 0.671) between DSAEK and PK at 1 (94% vs 90%), 2 (88% vs 85%), and 3 years (87% vs 85%). Compared with PK, DSAEK resulted in lower EC loss with comparable cumulative graft survival rates for up to 3 years in patients with Fuchs' dystrophy and BK.

Effects of metoprolol sustained-release tablets on vessel endothelial function and survival myocardium in hypertensive patients with stable angina pectoris

Objective To explore the effect of metoprolol sustained-release tablets on vessel endothelial function and survival myocardium in hypertensive patients with stable angina pectoris.Methods 182 patients with hypertension who had received coronary angiography were involved in the study.According to the results of angiography ( coronary stenosis=50％ ),the patients were divided into three groups:single-vessel disease group ( single-vessel group,n=47 ),double-vessel disease group ( double-vessel group,n =49 ),multi-vessel disease group ( multi-vessel group,n =44 ),and control group ( no coronary stenosis or ＜ 50％,n =42 ).After a 12-month treatment with conventional drugs plus metoprolol sustained-release tablets,the clinical presentations,laboratory findings,blood pressure,clinical features,echocardiography,and radionuclide ventriculography were measured and analyzed.Results As the degree of coronary artery lesions increased,FMD at hours 1 and 4 and 99Tcm-MIBI uptake fraction were worse in hypertensive patients with stable angina pectoris.12 months after using of metoprolol sustained-release tablets,a significant improvement in FMD at hours 1 and 4 and 99Tcm-MIBI uptake fraction were observed in more increased vessel coronary disease groups ( double-vessel group and multi-vessel group ),which increased ( 2.72 ± 0.51 and 4.20 ± 1.47 ),( 2.50 ±0.65 and 4.31±1.85 )and ( 0.54 ± 0.07 and 0.68 ± 0.16 ).Conclusions For hypertensive patients with stable angina pectoris,metoprolol sustained-release tablets can effectively improve vessel endothelial function and survival myocardium. Key words: Hypertension; Angina pectoris; Brachial artery; Survival myocardium; Metoprolol sustained-release tablets

Unraveling the influence of endothelial cell density on VEGF-A signaling

AbstractVascular endothelial growth factor-A (VEGF) is the master determinant for the activation of the angiogenic program leading to the formation of new blood vessels to sustain solid tumor growth and metastasis. VEGF specific binding to VEGF receptor-2 (VEGFR-2) triggers different signaling pathways, including phospholipase C-γ (PLC-γ) and Akt cascades, crucial for endothelial proliferation, permeability, and survival. By combining biologic experiments, theoretical insights, and mathematical modeling, we found that: (1) cell density influences VEGFR-2 protein level, as receptor number is 2-fold higher in long-confluent than in sparse cells; (2) cell density affects VEGFR-2 activation by reducing its affinity for VEGF in long-confluent cells; (3) despite reduced ligand-receptor affinity, high VEGF concentrations provide long-confluent cells with a larger amount of active receptors; (4) PLC-γ and Akt are not directly sensitive to cell density but simply transduce downstream the upstream difference in VEGFR-2 protein level and activation; and (5) the mathematical model correctly predicts the existence of at least one protein tyrosine phosphatase directly targeting PLC-γ and counteracting the receptor-mediated signal. Our data-based mathematical model quantitatively describes VEGF signaling in quiescent and angiogenic endothelium and is suitable to identify new molecular determinants and therapeutic targets.

Functional SNPs in vascular endothelial growth factor (VEGF-A) and overall survival (OS) in bevacizumab-treated patients with metastatic colorectal cancer (mCRC).

3594 Background: Functional SNPs in VEGF-A are prognostic for OS in several malignancies, but not described definitively in mCRC. In bevacizumab-treated patients with advanced breast cancer, VEGF -2578AA and -1154A were predictive of OS—the latter SNP reported in a recent mCRC series as well. We examined the association between five functional VEGF-A SNPs and OS in a large cohort of bevacizumab-treated patients with mCRC. Methods: 403 patients with mCRC treated from 2004-2010 were included in a retrospective analysis. DNA extraction, genotyping, and SNP evaluation were performed according to standard protocols. Survival was calculated from the time of Stage IV diagnosis until death. Data were censored as of 12/31/2010. Results: There were 279 deaths in this group of 403 patients (69%). Median age was 55.7 (24-86 y), and 54% of patients were male. Age, sex, race, tumor grade, chemotherapies, and curative surgery (metastatectomy to negative margins) were considered. Significant clinical predictors of OS in univariate Cox modeling were cetuximab treatment [HR=1.46; 95% CI 1.13-1.88; p=0.0014], irinotecan treatment [HR=2.10; 1.32-3.35; p<0.001], and curative surgery [HR=0.36; 0.25-0.75; p<0.001]. Significant negative interaction was found between both cetuximab and irinotecan and curative surgery, and in modeling that included this interaction, only surgery remained predictive. In multivariate analysis, no association was found between VEGF-A and OS (Table). Conclusions: There was no association between five functional VEGF-A SNPs and OS in this large bevacizumab-treated mCRC cohort. [Table: see text]

Abstract 4380: Effect of sunitinib on endothelial-pericyte cell interaction

Abstract Background: Sunitinib is a multitargeted tyrosine kinase receptor inhibitor, which has been used for the last 5 years as an anti-angiogenic drug for the treatment of metastasic renal cell carcinoma (RCC). Sunitinib treatment induces an initial favorable response in patients with RCC, however, resistant to sunitinib occurs in the majority of patients after ∼11 months of treatment. One of the mechanism that has been hypothesized to explain the resistance of RCC to sunitinib is an increase in pericyte cell coverage in the tumor vasculature. In vivo, pericytes and endothelial cells are in close proximity and they frequently have direct contact with each other through gap junctions. Endothelial-pericytes interactions regulate vascular development, endothelial cell permeability, vessel stabilization, vessel maturation, and remodeling. Moreover, pericytes have been demonstrated to secrete paracrine factors that stimulate signaling pathways implicated in endothelial cell differentiation and survival. Previous studies have shown that anti-angiogenic drugs may disrupt the physical interaction between endothelial cells and pericytes in vivo. Moreover, in vitro studies have demonstrated that the loss of endothelial-pericyte contact may decrease the level of expression of SMA and NG2, the two most important pericyte markers used in vivo. The aim of this study is to analyze the endothelial-pericyte cell interactions in vitro and the effect that sunitinib has in these interactions. Methods and Results: HUVEC (Human Umbilical Vein Endothelial Cells) and hPC-PL (Human Pericytes from Placenta) cells were utilized to characterize in vitro the effect of sunitinib in endothelial-pericyte cell interactions. The effect of sunitinib in cell proliferation of each primary culture was determined by using crystal violet and growth curve assays. Sunitinib, at 1uM concentration, decreases proliferation rate of endothelial and pericyte cells, process that was reversible after sunitinib withdrawal. Cocultures of HUVEC and hPC-PL cells showed that both cells types start to physically interact after 24 hours. After 48 hours of co-culture, hPC-PL cells in close proximity to endothelial cells overexpressed the pericyte markers NG2 and SMA, process that have been reported to be dependent on gap junction formation. Use of conditioned media obtained from endothelial cells did not induce overexpression of NG2 or SMA in hPC-PL cells, which suggests physical contact between pericyte and endothelial cells was necessary for pericyte to over-express NG2 and SMA. In addition, co-culture experiments performed in the presence of sunitinib did not affect the physical cell-to-cell interaction between HUVEC and hPC-PL. Conclusion: These results indicate endothelial-pericyte cell interactions are not only an important mechanism for endothelial survival signaling, but also are necessary to maintain pericyte differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4380. doi:1538-7445.AM2012-4380

Activated Pericytes and the Inhibition of Renal Vascular Stability

The hallmark feature of CKD and its progression to end stage is the presence and severity of interstitial fibrosis.1,2 This long-recognized association is the prime driving force of research to understand the nature of renal scarification. Much of the research in this area can be grouped into one of two broad categories: efforts geared toward understanding the biochemical and pathophysiological factors that govern the degree of progression and efforts aimed at defining the cellular and molecular elements that comprise renal scars. With regard to the former, it is clear that a profibrotic environment is promoted by activity from a variety of factors with activity driving the production of extracellular matrix (ECM). Of these, TGFβ and other profibrotic cytokines have been widely studied, and these are enhanced by local tissue injury, epithelial cell cycle arrest, cell differentiation status, or hypoxia.1,3

Endothelial microparticles: missing link in endothelial dysfunction?

Vascular homeostasis is an integral reflection of quiescent, but competent endothelium. Endothelial microparticles (EMPs) are circulating submicron-sized membranous vesicles released by endothelium that are increasingly recognized and play a multitude of biological roles reflecting competency of endothelial function and vascular biology. EMPs have been shown to act as primary and secondary messengers of vascular inflammation, thrombosis, vasomotor response, angiogenesis, and endothelial survival. These often-neglected vesicles are emerging as potentially useful indicators of dysfunctioning endothelium, whereby differential presence of EMPs is linked to disease manifestation. In this review, we outline the evolving understanding of EMPs from mere passive vesiculation to active messenger in pathophysiological responses. Current evidence implicating EMPs in cardiopulmonary, renal, cerebral, and metabolic disorders warrants vigorous clinical validation. While studies of EMPs face significant technical challenges that await standardization and consolidation, unravelling its biological significance in endothelial dysfunction could herald the advent of novel non-invasive surveillance platforms for vascular health, risk stratification, and disease prognostication.