Endothelial Survival Research Articles

Vascular endothelial growth factor and its relationship to the prognosis and treatment of breast, ovarian, and cervical cancer

Tumor neovascularization is a complex process that plays a crucial role in the development of many different types of cancer. Vascular endothelial growth factor (VEGF) is a potent mitogen that is involved with mitogenesis, angiogenesis, endothelial survival, and the induction of hematopoiesis. By increasing vascular permeability in endothelial cells, it helps tumors recruit wound-healing proteins fibrin and fibrinogen from the plasma, suggesting that tumor formation is a process of abnormal wound healing dependent on the ability to generate a blood supply. The human female reproductive tract is highly dependent on VEGF for normal functions such as endometrial proliferation and development of the corpus luteum. The unique influence of female sex steroid hormones on the expression and activity of VEGF deems angiogenesis an important facet of the development of breast and ovarian cancer. Additionally, the up-regulation of VEGF by the E6 oncoprotein of the human papillomavirus suggests that VEGF plays an important role in the development of cervical cancer. Clinical trials have investigated the humanized monoclonal antibody bevacizumab as potential treatment for all three forms of cancer; the data show that in breast cancer, the use of bevacizumab may lengthen the disease-free survival for women with advanced breast cancer, but does not appear to change their overall survival. It may have a role as salvage chemotherapy for ovarian and cervical cancer, though further research is needed to establish it as a definitive form of treatment.

Safety of DSAEK in Patients With Previous Glaucoma Filtering Surgery

To report 4 cases of Descemet stripping automated endothelial keratoplasty (DSAEK) in the presence of previous glaucoma filtering surgery. Observational case series. Review of clinical data of 4 patients who underwent DSAEK successfully performed in the presence of previous glaucoma filtering surgery with endothelial survival rates comparable with larger series published previously and good postoperative intraocular pressure (IOP) control. The endothelial cell loss was 36% and 39% mean cell loss at 6 months and 1 year postoperatively. The IOP remained well controlled within target levels in all patients. No complications were reported in any of the 4 cases. Corneal endothelial failure can be successfully managed with DSAEK in glaucoma patients with previous filtering surgery with good endothelial survival rates and IOP control.

Cigarette smoke regulates VEGFR2-mediated survival signaling in rat lungs

BackgroundVascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2)-mediated survival signaling is critical to endothelial cell survival, maintenance of the vasculature and alveolar structure and regeneration of lung tissue. Reduced VEGF and VEGFR2 expression in emphysematous lungs has been linked to increased endothelial cell death and vascular regression. Previously, we have shown that CS down-regulated the VEGFR2 and its downstream signaling in mouse lungs. However, the VEGFR2-mediated survival signaling in response to oxidants/cigarette smoke (CS) is not known. We hypothesized that CS exposure leads to disruption of VEGFR2-mediated endothelial survival signaling in rat lungs.MethodsAdult male Sprague-Dawley rats were exposed CS for 3 days, 8 weeks and 6 months to investigate the effect of CS on VEGFR2-mediated survival signaling by measuring the Akt/PI3-kinase/eNOS downstream signaling in rat lungs.Results and DiscussionWe show that CS disrupts VEGFR2/PI3-kinase association leading to decreased Akt and eNOS phosphorylation. This may further alter the phosphorylation of the pro-apoptotic protein Bad and increase the Bad/Bcl-xl association. However, this was not associated with a significant lung cell death as evidenced by active caspase-3 levels. These data suggest that although CS altered the VEGFR2-mediated survival signaling in the rat lungs, but it was not sufficient to cause lung cell death.ConclusionThe rat lungs exposed to CS in acute, sub-chronic and chronic levels may be representative of smokers where survival signaling is altered but was not associated with lung cell death whereas emphysema is known to be associated with lung cell apoptosis.

Descemet's Stripping Automated Endothelial Keratoplasty Outcomes Compared with Penetrating Keratoplasty from the Cornea Donor Study

To assess outcomes 1 year after Descemet's stripping automated endothelial keratoplasty (DSAEK) in comparison with penetrating keratoplasty (PKP) from the Specular Microscopy Ancillary Study (SMAS) of the Cornea Donor Study. Multicenter, prospective, nonrandomized clinical trial. A total of 173 subjects undergoing DSAEK for a moderate risk condition (principally Fuchs' dystrophy or pseudophakic/aphakic corneal edema) compared with 410 subjects undergoing PKP from the SMAS who had clear grafts with at least 1 postoperative specular image within a 15-month follow-up period. The DSAEK procedures were performed by 2 experienced surgeons per their individual techniques, using the same donor and similar recipient criteria as for the PKP procedures in the SMAS performed by 68 surgeons at 45 sites, with donors provided from 31 eye banks. Graft success and complications for the DSAEK group were assessed and compared with the SMAS group. Endothelial cell density (ECD) was determined from baseline donor, 6-month (range, 5-7 months), and 12-month (range, 9-15 months) postoperative central endothelial images by the same reading center used in the SMAS. Endothelial cell density and graft survival at 1 year. Although the DSAEK recipient group criteria were similar to the PKP group, Fuchs' dystrophy was more prevalent in the DSAEK group (85% vs. 64%) and pseudophakic corneal edema was less prevalent (13% vs. 32%, P<0.001). The regraft rate within 15 months was 2.3% (DSAEK group) and 1.3% (PKP group) (P = 0.50). Percent endothelial cell loss was 34+/-22% versus 11+/-20% (6 months) and 38+/-22% versus 20+/-23% (12 months) in the DSAEK and PKP groups, respectively (both P<0.001). Preoperative diagnosis affected endothelial cell loss over time; in the PKP group, the subjects with pseudophakic/aphakic corneal edema experienced significantly higher 12-month cell loss than the subjects with Fuchs' dystrophy (28% vs. 16%, P = 0.01), whereas in the DSAEK group, the 12-month cell loss was comparable for the 2 diagnoses (41% vs. 37%, P = 0.59). One year post-transplantation, overall graft success was comparable for DSAEK and PKP procedures and endothelial cell loss was higher with DSAEK.

Role of Nrf2 in retinal vascular development and the vaso-obliterative phase of oxygen-induced retinopathy

In the initial stage of retinopathy of prematurity (ROP), hyperoxia causes retinal blood vessel obliteration. This is thought to occur in part through oxidative stress-induced apoptosis of endothelial cells. This study was designed to determine what role NF-E2-related factor 2 (Nrf2) plays in this process. Nrf2 is a transcription factor of the anti-oxidant response element that, if induced, may protect the retina from hyperoxia-induced oxidative stress. Nrf2 knockout mice (Nrf2−/−), Nrf2 wild type control mice (Nrf2+/+), and C57BL/6 mice were exposed to hyperoxia (75% O 2) or normoxia from P7 through P12. Mice were sacrificed on P9 and P12 and the retinas were stained with GSA lectin-Cy3 to visualize retinal blood vessels. Hyperoxia exposed retinas were flat mounted and photographed, then the size of the avascular areas was determined. Additionally, retinas were cryopreserved after lectin staining and area analysis and then sectioned. Secondary or deep capillaries were then hand-counted in sections. In hyperoxia-treated mice, the avascular areas in Nrf2−/− P9 mice were significantly larger than those in Nrf2+/+ P9 mice ( P = 0.01). However, there was no significant difference between Nrf2−/− and Nrf2+/+ mice at P12. Avascular areas at P12 were significantly smaller than that at P9 in Nrf2−/−, Nrf2+/+, and C57BL/6 mice ( P = 0.0011, P = 0.009, and P = 0.001 respectively). The numbers of deep or secondary capillaries in air-reared Nrf2−/− mice were significantly decreased, when compared to Nrf2+/+ mice at P9 ( P = 0.0082). On the other hand, there was no significant difference in deep capillary formation between air-reared Nrf2−/− and Nrf2+/+ mice at P12. Akt signaling activates Nrf2 and Akt was localized to retinal blood vessels in all animals and was increased in Nrf2+/+ and Nrf2−/− mice exposed to hyperoxia as compared to normoxia mice. Interestingly, during normal development this protection by Nrf2 occurs in a specific window of time that is also shared by angiogenesis. Hyperoxia treatment revealed a similar window of time where Nrf2 regulated anti-oxidant production was beneficial and contributed to the endothelial survival.

Descemet's Membrane Endothelial Keratoplasty: Prospective Multicenter Study of Visual and Refractive Outcomes and Endothelial Survival

Descemet's Membrane Endothelial Keratoplasty: Prospective Multicenter Study of Visual and Refractive Outcomes and Endothelial Survival

Role of Vascular Endothelial Growth Factor in Kidney Disease

Vascular endothelial growth factor (VEGF) is a main regulator of blood vessel growth and plays an important role in promoting endothelial survival and maintaining the microvasculature. The kidney is a highly vascularized organ and has two important microvasculatures; glomerular and peritubular capillaries. Loss of these capillaries is strongly associated with the progression of chronic kidney disease (CKD) to end-stage renal disease. In several kidney disease animal models, VEGF expression in the kidney is decreased and administration of VEGF is protective. Recent clinical observations revealed that blocking VEGF by endogenous inhibitor (soluble Flt-1) in preeclampsia and monoclonal antibody against VEGF in cancer patients cause proteinuria and renal dysfunction. However, plasma VEGF levels in diabetic nephropathy patients are increased and blocking VEGF improved diabetic nephropathy in animal models. Increased plasma VEGF levels have been reported in CKD patients. Deleterious effects of VEGF have been demonstrated in atherosclerosis and sepsis, which are frequent complications in CKD patients. Although administrating VEGF or novel drugs that activate VEGF pathway may improve the progression of CKD, careful monitoring will be required when CKD patients have complications of diabetes, atherosclerosis or sepsis.

Histone Deacetylase 3 Is Critical in Endothelial Survival and Atherosclerosis Development in Response to Disturbed Flow

Histone deacetylase 3 (HDAC3) is known to play a crucial role in the differentiation of endothelial progenitors. The role of HDAC3 in mature endothelial cells, however, is not well understood. Here, we investigated the function of HDAC3 in preserving endothelial integrity in areas of disturbed blood flow, ie, bifurcation areas prone to atherosclerosis development. En face staining of aortas from apolipoprotein E-knockout mice revealed increased expression of HDAC3, specifically in these branching areas in vivo, whereas rapid upregulation of HDAC3 protein was observed in endothelial cells exposed to disturbed flow in vitro. Interestingly, phosphorylation of HDAC3 at serine/threonine was observed in these cells, suggesting that disturbed flow leads to posttranscriptional modification and stabilization of the HDAC3 protein. Coimmunoprecipitation experiments showed that HDAC3 and Akt form a complex. Using a series of constructs harboring deletions, we found residues 136 to 206 of HDAC3 to be crucial in this interaction. Enforced expression of HDAC3 resulted in increased phosphorylation of Akt and upregulation of its kinase activity. In line with these findings, knockdown of HDAC3 with lentiviral vectors (shHDAC3) led to a dramatic decrease in cell survival accompanied by apoptosis in endothelial cells. In aortic isografts of apolipoprotein E-knockout mice treated with shHDAC3, a robust atherosclerotic lesion was formed. Surprisingly, 3 of the 8 mice that received shHDAC3-infected grafts died within 2 days after the operation. Miller staining of the isografts revealed disruption of the basement membrane and rupture of the vessel. Our findings demonstrated that HDAC3 serves as an essential prosurvival molecule with a critical role in maintaining the endothelial integrity via Akt activation and that severe atherosclerosis and vessel rupture in isografted vessels of apolipoprotein E-knockout mice occur when HDAC3 is knocked down.

Safety of Bevacizumab in Advanced Ovarian and Müllerian Cancers: A Review

Abstract Despite the armamentarium of cytotoxic agents available in the treatment of patients with advanced recurrent ovarian, fallopian, and peritoneal disease, enthusiasm for these agents has been tempered by their toxicity profile, poor response rate, and eventually, the emergence of resistance. In recent years, focus has shifted to an understanding of signaling pathways that promote angiogenesis. Tumor angiogenesis is well established as essential for the growth and metastasis of solid tumors. This process involves the recruitment of mature vasculature and circulating endothelial cells and proangiogenic soluble mediators, one of which includes the vascular endothelial growth factor (VEGF). This factor has several known activities, such as mitogenesis, angiogenesis, endothelial survival, enhancement of vascular permeability, and effects on hemodynamic status. Vascular endothelial growth factor is a 45-kDA glycoprotein that is regulated by oxygen tension and also by various growth factors, hormones, and oncogenes. Inhibiting VEGF and its action has been the focus of intense research in several disciplines and has emerged as an attractive focus for the design of targeted therapeutics in the field of ovarian cancer. Numerous agents targeting angiogenesis are currently being explored. These include monoclonal antibodies to the VEGF ligand, small tyrosine kinase inhibitors that target the vascular endothelial receptor, and soluble decoy VEGF receptors. The most studied agent to date has been bevacizumab, a recombinant humanized monoclonal antibody to the VEGF ligand. Although the efficacy of this agent has been well established in multiple disciplines, several concerns have been raised as to the unique adverse effects of this agent.

Activated Protein C and Acute Kidney Injury: Selective Targeting of PAR-1

Protein C is a plasma serine protease that when activated plays a central role in modulating the function of the vascular endothelium and its interface with the innate immune system. Activated protein C (APC) has a dual mechanism of action via the feedback inhibition of thrombin generation, and as an agonist of protease activated receptor-1 (PAR-1). Through different cofactor interactions, this dual mechanism of antithrombotic and cytoprotective activity results in the ability of APC to modulate endothelial dysfunction by blocking cytokine signaling, functional cell adhesion expression, vascular permeability, apoptosis, and modulating leukocyte migration and adhesion. Deficiency in protein C, which occurs during systemic inflammatory activation, is highly associated with organ dysfunction. APC has shown efficacy in a number of preclinical models of thrombosis and ischemia, and the recombinant human APC drotrecogin alfa (activated), reduces mortality in patients with high-risk severe sepsis. The ability of APC to suppress pro-inflammatory pathways and enhance cellular survival suggests that APC plays a key role in the adaptive response to protect the vessel wall from insult and to enhance endothelial, cellular, and organ survival. The focus of this review will be to summarize the emerging data suggesting the potential therapeutic benefit of APC and related members of the pathway in the prevention and treatment of acute kidney injury.

Hypoxia-Inducible Factor-1 in Multiple Myeloma Progression.

Abstract 1812Poster Board I-838Multiple Myeloma (MM) is a malignancy of immunoglobulin (Ig)-synthesizing plasma cells, that home to and expand in the bone marrow. Similarly to other tumours its development is correlated to the formation of regions of hypoxia, which may be a prognostic indicator and determinant of malignant progression. It is known how in solid tumours the degree of intra-tumoral hypoxia is positively correlated with the expression of the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 is composed of HIF-1á and HIF-1β subunits and its production has been identified as a key element in allowing cells to adapt and survive in a hostile hypoxic environment via a variety of pathways. In hypoxia conditions, the HIF-1á subunit becomes stable and regulates the expression of target genes. When activated HIF-1á also targets those genes which are required for angiogenesis, the development of new blood vessels from an existing vascular network. Angiogenesis represents a constant hallmark of MM progression. In response to hypoxia plasma cells and stromal cells (endothelial cells [ECs], macrophages, mast cells) within the tumour express Vascular Endothelial Growth Factor (VEGF), a mitogen and survival factor specific for endothelial cells. VEGF is the major regulator of tumor-associated angiogenesis. HIF-1á directly activates transcription of the VEGF gene and this leads to autocrine signal transduction that is critical for angiogenesis.In this study we demonstrate the role of HIF-1á in MM angiogenesis. The constitutive stabilization of HIF-1á contributes to increase angiogenesis in MM. Our data show that HIF-1á is stabilized in the nucleus of MM endothelial cells (MMECs) but not in ECs of Monoclonal Gammopathies of Undetermined Significance (MGECs) and in Human Umbilical Vein ECs (HUVECs) used as controls. Western Blot and Enzyme-Linked Immunosorbent Assay (ELISA) analyses show the overexpression of HIF-1á and the proteic products of its target genes VEGF and VEGF Receptor (VEGFR)-1, in patients with relapsed disease and in MM progression but not in patients with nonactive MM (avascular phase). Moreover, immunofluorescent staining confirm the nuclear stabilization of HIF-1á in MMECs.At mRNA level all ECs express same quantity of HIF-1á mRNA, as confirmed by RT-PCR and Real-time RT-PCR, indicating that in MMECs the post-trascriptional control is affected. Finally, we show that the inhibition of HIF-1á by siRNA suppresses vessel formation in vitro and promote ECs apoptosis. Our findings indicate that HIF-1á plays an important role in MM progression and that it is correlated to the angiogenic switch from nonactive MM to active MM. Furthermore these data suggest that HIF-1á may represent a target for the MM antiangiogenic treatment. DisclosuresNo relevant conflicts of interest to declare.

Silencing of BDNF Expression by shRNA Down-Regulates Stroma-Derived VEGF Secretion and Inhibits In Vivo Multiple Myeloma Growth and Angiogenesis in the Bone Marrow Milieu.

Abstract 423 Introduction:Bone marrow (BM) neovascularization and vascular endothelial growth factor (VEGF) expression in multiple myeloma (MM) correlate with disease progression and poor prognosis. Brain derived neurotrophic factor (BDNF) is highly expressed by malignant plasma cells isolated from MM patients. Recently, BDNF is identified as an endothelial survival factor to induce angiogenesis and increase levels of VEGFR in tyrosine receptor kinase B (TrkB)-positive endothelial cells. In addition, BDNF participates in promoting vessel formation by human umbilical vein endothelial cells. High levels of BDNF are detected in the serum of patients with active MM and comparable VEGF levels. Since BDNF and its specific receptor-TrkB are expressed by stromal cells within the bone marrow microenvironment, we sought to evaluate the involvement of BDNF-TrkB axis in myeloma-marrow stroma interaction and its effects on BM neovascularization by regulating VEGF expression. Methods and results: BM stromal cells (BMSCs) isolated from MM patients (n=11) and control health donors (n=8) were confirmed to express BDNF high-affinity specific receptor TrkB by real-time RT-PCR and immunoblotting. Stimulation of BMSCs with exogenous BDNF induced a time- and dose- dependent increase in VEGF secretion (1.8- to 3.3- fold, p<0.01), which was completely abolished by K252α, an inhibitor of TrkB. Western blot analysis demonstrated that exposing BMSCs with BDNF for 30 min at varying concentration from 25 ng/mL to 150 ng/mL leaded to a dose-dependent phosphorylation of signal transducers and activators of transcription 3 (STAT3). Nuclear translocation and DNA-binding of STAT3 was further confirmed by Electrophoretic mobility shift assay (EMSA), BDNF induced a 2.6-fold increase of STAT3/DNA complex in BMSCs (100 ng/mL, 60min). Inhibition of STAT3, a downstream signaling molecule of TrKB by AG490 eliminated stimulating effects of BDNF on BMSCs as well. Meanwhile, RPMI 8226 cells were transfected with lentiviral based shBDNF-pGCSIL-GFP or control vector. Pure population of transfected RPMI 8226 cells were firstly isolated by Fluorescence Activated Cell Sorting (FACS), and then specific BDNF knockdown was double verified by real-time RT-PCR and western blot. BMSCs were co-cultured with BDNF shRNA RPMI 8226 cells in a non-contact transwell system. VEGF concentration in supernatants of BMSCs and BDNF shRNA RPMI 8226 cell co-cultures were significantly decreased when compared with control group, in which BMSCs were cocultured with control RPMI 8226 cells (p<0.05). STAT3 phosphorylation was also inhibited in BMSCs co-cultured with BDNF shRNA RPMI 8226 cells(p<0.01). Furthermore, BDNF shRNA RPMI 8226 cells were inoculated subcutaneously with BMSCs in irradiated nude mice.The tumor volume was measured every three days by in-vivo optical imaging and capliper. In addition, Microvessel density (MVD) was assessed by immunohistochemical analysis for CD31 expression. In vivo results demonstrated that silencing of MM derived BDNF expression significantly inhibited angiogenesis and VEGF expression in the BM microenvironment. Moreover, suppression of BDNF inhibited MM tumor growth and prolonged survival in mice. Conclusion:Our studies demonstrate that BDNF, as a potential stimulator of angiogenesis, contributes to MM tumorgenesis; it mediates stromal–MM cell interactions via selective activation of specific tyrosine kinase receptors TrkB and downstream signal transducer STAT3 in regulating VEGF secretion. Disclosures:No relevant conflicts of interest to declare.

Descemet's Membrane Endothelial Keratoplasty: Prospective Multicenter Study of Visual and Refractive Outcomes and Endothelial Survival

To describe Descemet's membrane endothelial keratoplasty (DMEK) techniques, perioperative challenges, management, and visual and refractive outcomes. Prospective, multicenter, consecutive case series. Sixty eyes of 56 patients with Fuchs' endothelial dystrophy, pseudophakic bullous keratopathy, or failed previous graft. Descemet's membrane (DM) and endothelium were stripped from donor corneas submerged in corneal storage solution in a corneal viewing chamber. Donor DM diameters were 8.5 or 9.0 mm. The central 7 mm of DM was stripped from the recipient cornea. After staining with trypan blue to improve visualization, donor DM was inserted through a 2.8-mm incision. Descemet's membrane endothelial keratoplasty was performed alone (n = 48) or was combined with phacoemulsification and lens implantation (n = 11), pars plana vitrectomy (n = 2), or both. Best spectacle-corrected visual acuity (BSCVA), manifest refraction, and endothelial cell density. Median BSCVA was 20/30 at 1 month (range, 20/20-20/60), improving from 20/50 (range, 20/25-hand movements) before DMEK, excluding 4 eyes (7%) with preexisting ocular pathologic features that limited visual potential. At 3 months, 26% had 20/20 vision, 63% saw 20/25 or better, and 94% saw 20/40 or better. Refractive cylinder remained unchanged at 0.9 diopters (D; P = 0.93), and a hyperopic shift of 0.49+/-0.63 D (P = 0.0091) was noted in DMEK single procedures. Endothelial cell loss was 30%+/-20% at 3 months and 32%+/-20% in 38 eyes that reached the 6-month examination. Median pachymetry decreased from 660 mum before surgery to 530 mum. Descemet's membrane stripped successfully from 60 of 72 donor corneas; 6 were converted successfully to Descemet's stripping automated endothelial keratoplasty (DSAEK) and 6 (8%) were discarded. Only 1 graft detached completely, but air was reinjected in 38 eyes (63%), mainly for partial detachments. Five DMEK corneas (8%) failed to clear and were replaced successfully with DMEK or DSAEK. All remained clear at last follow-up. Compared with DSAEK, DMEK provided a significantly higher rate of 20/20 and 20/25 vision, with comparable endothelial cell loss. Descemet's membrane endothelial keratoplasty restored physiologic pachymetry, but donor preparation and attachment currently are more challenging than with DSAEK.

Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) lacking expression of steroid receptors and human epidermal growth factor receptor 2, having chemotherapy as the only therapeutic option, is characterised by early relapses and poor outcome. We investigated intratumoural (i.t.) levels of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and survival in patients with TNBC compared with non-TNBC. Patients and methodsVEGF levels were determined by an enzyme immunosorbent assay in a retrospective series consisting of 679 consecutive primary breast cancer patients. ResultsEighty-seven patients (13%) were classified as TNBC and had significantly higher VEGF levels; median value in TNBC was 8.2 pg/μg DNA compared with 2.7 pg/μg DNA in non-TNBC (P<0.001). Patients with TNBC had statistically significant shorter recurrence-free survival [hazard ratio (HR)=1.8; P=0.0023], breast cancer-corrected survival (HR=2.2; P=0.004) and overall survival (HR=1.8; P=0.005) compared with non-TNBC. Patients with TNBC relapsed earlier than non-TNBC; mean time from diagnosis to first relapse was 18.8 and 30.7 months, respectively. The time between first relapse and death was also shorter in TNBC: 7.5 months versus 17.5 months in non-TNBC (P=0.087). ConclusionsOur results show that TNBC have higher i.t. VEGF levels compared with non-TNBC. Ongoing clinical trials will answer if therapy directed towards angiogenesis may be an alternative way to improve outcome in this poor prognosis group.

The angiopietin-1–Tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice

The role of the angiopoietin-1 (Ang1)–Tie2 pathway in the pathogenesis of pulmonary arterial hypertension (PAH) is controversial. Although Ang1 is well known to prevent endothelial activation and injury in systemic vascular beds, this pathway has been suggested to mediate pulmonary vascular remodeling in PAH. Therefore, we used transgenic models to determine the effect of increased or decreased Tie2 activity on the development of PAH. We now report modest spontaneous elevation in right ventricular systolic pressure in Tie2-deficient mice (Tie2+/−) compared with wild-type (WT) littermate controls, which was exacerbated upon chronic exposure to the clinically relevant PAH triggers, serotonin (5-HT) or interleukin-6 (IL-6). Moreover, overexpression of Ang1 in transgenic mice had no deleterious effect on pulmonary hemodynamics and, if anything, blunted the response to 5-HT. Exposure to 5-HT or IL-6 also decreased lung Ang1 expression, further reducing Tie2 activity and inducing pulmonary apoptosis in the Tie2+/− group only. Similarly, cultured pulmonary artery endothelial cells subjected to Tie2 silencing demonstrated increased susceptibility to apoptosis after 5-HT treatment. Finally, treatment of Tie2-deficient mice with Z-VAD, a pan-caspase inhibitor, prevented the pulmonary hypertensive response to 5-HT. Thus, these findings firmly establish that endothelial survival signaling via the Ang1–Tie2 pathway is protective in PAH.

Vascular endothelial growth factor enhances migration of astroglial cells in subventricular zone neurosphere cultures

Vascular endothelial growth factor (VEGF) is an endothelial and neuronal survival factor and a mitogen for endothelial cells and astrocytes in both explant and in vivo injury models. In the CNS, interplay between the vasculature and neural stem progenitor (NSP) cells is required for the maintenance of angiogenic/neurogenic coordination in the germinal niche in the subventricular zone (SVZ) of the lateral ventricle. Using an in vitro SVZ neurosphere (NS) model, this study aimed to understand the direct effects of VEGF and its receptor signaling on neonatal NSP cell growth and migration. Our data indicate that VEGF administration, compared with untreated or brain-derived neurotrophic factor-treated NS, significantly increased growth and migratory capacity of glial fibrillary acidic protein (GFAP)(+) and nestin(+) NSP cells and in secondary cultures induced a stellate astrocyte morphology. Blockade of both VEGF, which is normally expressed in some NS cells, and its flt-1 receptor signaling by neutralizing antibodies caused morphological changes specifically in GFAP(+) cells and disrupted sphere formation and outward migration. These cells did not appear as conventional polygonal astrocytes; their process growth was severely restricted, and overall migration was reduced by up to 76% of control cultures. Blockade of VEGF's flk-1 receptor reduced VEGF expression and caused a lesser, though significant, decrease (29%) in NSP (GFAP(+)) cell migration. The results show that both VEGF and, in particular, flt-1 receptor signaling are critical to the proper configuration of the NS and its subsequent development. VEGF is also an important growth and migratory factor particularly for GFAP(+) cells developing in SVZ-derived NS in culture.

Endothelial Cells Support Survival, Proliferation, and Neuronal Differentiation of Transplanted Adult Ischemia-Induced Neural Stem/Progenitor Cells After Cerebral Infarction

Transplantation of neural stem cells (NSCs) has been proposed as a therapy for a range of neurological disorders. To realize the potential of this approach, it is essential to control survival, proliferation, migration, and differentiation of NSCs after transplantation. NSCs are regulated in vivo, at least in part, by their specialized microenvironment or "niche." In the adult central nervous system, neurogenic regions, such as the subventricular and subgranular zones, include NSCs residing in a vascular niche with endothelial cells. Although there is accumulating evidence that endothelial cells promote proliferation of NSCs in vitro, there is no description of their impact on transplanted NSCs. In this study, we grafted cortex-derived stroke-induced neural stem/progenitor cells, obtained from adult mice, onto poststroke cortex in the presence or absence of endothelial cells, and compared survival, proliferation, and neuronal differentiation of the neural precursors in vivo. Cotransplantation of endothelial cells and neural stem/progenitor cells increased survival and proliferation of ischemia-induced neural stem/progenitor cells and also accelerated neuronal differentiation compared with transplantation of neural precursors alone. These data indicate that reconstitution of elements in the vascular niche enhances transplantation of adult neural progenitor cells.

Endothelial Keratoplasty: Vision, Endothelial Survival, and Complications in a Comparative Case Series of Fellows vs Attending Surgeons

To compare a 6-month postoperative vision, endothelial cell loss, and immediate postoperative complications in Descemet stripping automated endothelial keratoplasty (DSAEK) cases performed by an experienced corneal surgeon and his inexperienced fellows using an established technique. Retrospective analysis of prospectively gathered data in 327 consecutive DSAEK cases. DSAEK cases performed by fellows vs attending surgeons during a concurrent period were compared for 6-month postoperative best spectacle-corrected visual acuity (BSCVA), endothelial cell loss, and immediate postoperative complications. This series represents the initial consecutive cases performed by 3 cornea fellows using a technique identical to that used by the attending. After DSAEK, average BSCVA improved from 20/80 to 20/37 in the attending cases and 20/74 to 20/36 in the fellow cases. There was no statistical difference in endothelial cell loss between groups (32% vs 35%). The dislocation rate was 2% in the attending group and 1% in the fellow group. There were no primary graft failures or cases of pupillary block in either group. This study demonstrates no difference in vision or endothelial cell loss after DSAEK when performed by an experienced corneal surgeon or inexperienced fellows. Postoperative complications also were similar. Results and complications of DSAEK performed by supervised novice fellows can equal those of an experienced DSAEK surgeon.

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.

TGF-β Is Required for Vascular Barrier Function, Endothelial Survival and Homeostasis of the Adult Microvasculature

Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-β signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-β signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β signaling in vasoproliferative and vascular degenerative retinal diseases.