Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

Endothelial nitric oxide synthase is not essential for the development of fibrosis and portal hypertension in bile duct ligated mice

BACKGROUND/AIMS: It is postulated that nitric oxide (NO) is responsible for the hyperdynamic circulation of portal hypertension. Therefore, we investigated induction of fibrosis and hyperdynamic circulation in endothelial NO synthase knock-out (KO) mice. METHODS: Fibrosis was induced by bile duct ligation. Hemodynamic studies were performed after portal vein ligation. All studies were performed in wild-type (WT) and KO mice. RESULTS: Three to 4 weeks after bile duct ligation (BDL), both WT and KO groups had similar degrees of portal hypertension, 12 (9-14) and 11(8-15) mmHg, median (range), and liver function. Fibrosis increased from 0.0% in sham operated to 1.0 and 1.1% in WT and KO mice, respectively. Cardiac output was similar after portal vein ligation (20 and 17 ml/min in WT and KO mice, respectively). There was no difference in liver of mRNA for endothelin 1, inducible NO synthase (iNOS) and hem-oxygenase 1 (HO1); proteins of iNOS, HO1 and HO2; nor in endothelin A and B (EtA and EtB) receptor density between WT and KO mice after BDL. CONCLUSIONS: These results suggest that endothelial NO synthase is neither essential for the development of fibrosis and portal hypertension in bile duct ligated mice, nor for the hyperdynamic circulation associated with portal hypertension in the portal vein ligated mice.

Vascular dysfunction and reduced circulating endothelial progenitor cells in young healthy UK South Asian men

OBJECTIVES: The objective of this study was to examine determinants of excess coronary artery disease risk in UK South Asians, more prevalent in this population than UK Caucasians, by examining differences in risk factors, vascular function, and endothelial progenitor cells (EPCs). METHODS AND RESULTS: 24 South Asian and 25 Caucasian healthy age-matched nonsmoking men were studied. Vascular function was assessed by flow-mediated and GTN brachial artery dilatation and blood flow responses to infusion of ACh, SNP, and L-NMMA. EPC number and function were measured by flow cytometry (CD34, CD133, and KDR positive cells), and CFU/migration assays. Traditional risk factors and anthropometric measurements were similar in the groups. South Asians had higher fasting insulin levels (6.01 versus 3.62 microU/mL; P = 0.02). South Asians had lower FMD (6.9 versus 8.5%; P = 0.003), L-NMMA response (0.8 versus 1.3 mL/min/100 mL; P = 0.03), mean SNP response (9.5+/-0.6 versus 11.6+/-0.6; P = 0.02), EPC number (0.046+/-0.005% versus 0.085+/-0.009%; P = < 0.001), and CFU ability (CFU 4.29+/-1.57 versus 18.86+/-4.00; P = 0.005). EPC number was the strongest predictor of FMD. Ethnicity was the strongest predictor of EPC number. CONCLUSIONS: Healthy South Asian men are more insulin resistant, and demonstrate endothelial dysfunction and reduced EPC number and function compared with Caucasians. These abnormalities may contribute to their increased CAD risk.

VE-PTP maintains the endothelial barrier via plakoglobin and becomes dissociated from VE-cadherin by leukocytes and by VEGF

We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)-cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885-4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin-mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor alpha-activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, beta-catenin, and plakoglobin. Surprisingly, only plakoglobin but not beta-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not beta-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not beta-catenin.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Tenomodulin is necessary for tenocyte proliferation and tendon maturation

Tenomodulin (Tnmd) is a member of a new family of type II transmembrane glycoproteins. It is predominantly expressed in tendons, ligaments, and eyes, whereas the only other family member, chondromodulin I (ChM-I), is highly expressed in cartilage and at lower levels in the eye and thymus. The C-terminal extracellular domains of both proteins were shown to modulate endothelial-cell proliferation and tube formation in vitro and in vivo. We analyzed Tnmd function in vivo and provide evidence that Tnmd is processed in vivo and that the proteolytically cleaved C-terminal domain can be found in tendon extracts. Loss of Tnmd expression in gene targeted mice abated tenocyte proliferation and led to a reduced tenocyte density. The deposited amounts of extracellular matrix proteins, including collagen types I, II, III, and VI and decorin, lumican, aggrecan, and matrilin-2, were not affected, but the calibers of collagen fibrils varied significantly and exhibited increased maximal diameters. Tnmd-deficient mice did not have changes in tendon vessel density, and mice lacking both Tnmd and ChM-I had normal retinal vascularization and neovascularization after oxygen-induced retinopathy. These results suggest that Tnmd is a regulator of tenocyte proliferation and is involved in collagen fibril maturation but do not confirm an in vivo involvement of Tnmd in angiogenesis.

Dextran sulfate modulates MAP kinase signaling and reduces endothelial injury in a rat aortic clamping model

OBJECTIVE: Mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK1/2, noticeably influence ischemia/reperfusion injury (IRI). The complement inhibitor dextran sulfate (DXS) associates with damaged endothelium denudated of its heparan sulfate proteoglycan (HSPG) layer. Other glycosaminoglycan analogs are known to influence MAPK signaling. Hypothetically therefore, targeted intravascular cytoprotection by DXS may function in part through influencing MAPK activation to reduce IRI-induced damage of the vasculature. METHODS: IRI of the infrarenal aorta of male Wistar rats was induced by 90 minutes clamping followed by 120 minutes reperfusion. DXS (5 mg/mL) or physiologic saline (NaCl controls) was infused locally into the ischemic aortic segment immediately prior to reperfusion. Ninety minutes ischemia-only and heparinase infusion (maximal damage) experiments, as well as native rat aorta, served as controls. Aortas were excised following termination of the experiments for further analysis. RESULTS: DXS significantly inhibited IRI-induced JNK and ERK1/2 activation (P = .043; P =.005) without influencing the p38 pathway (P =.110). Reduced aortic injury, with significant inhibition of apoptosis (P = .032 for DXS vs NaCl), correlated with decreased nuclear factor kappaB translocation within the aortic wall. DXS treatment clearly reduced C1q, C4b/c, C3b/c, and C9 complement deposition, whilst preserving endothelial cell integrity and reducing reperfusion-induced HSPG shedding. Protection was associated with binding of fluorescein labeled DXS to ischemically damaged tissue. CONCLUSIONS: Local application of DXS into ischemic vasculature immediately prior to reperfusion reduces complement deposition and preserves endothelial integrity, partially through modulating activation of MAPKs and may offer a new approach to tackle IRI in vascular surgical procedures. CLINICAL RELEVANCE: The purpose of the present study was to determine the role of dextran sulfate (DXS), a glycosaminoglycan analog and complement inhibitor, in modulating intracellular MAPK signaling pathways, reducing complement activation and ultimately attenuating ischemia/reperfusion injury (IRI) in a rat aortic-clamping model, in part a surrogate model to study the microvasculature. The study shows a role for DXS in ameliorating endothelial injury by reducing IRI-mediated damage and intravascular, local inflammation in the affected aortic segment. DXS may be envisaged as an endothelial protectant in vascular injury, such as occurs during vascular surgical procedures.

The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction

The central nervous system (CNS) is tightly sealed from the changeable milieu of blood by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB). While the BBB is considered to be localized at the level of the endothelial cells within CNS microvessels, the BCSFB is established by choroid plexus epithelial cells. The BBB inhibits the free paracellular diffusion of water-soluble molecules by an elaborate network of complex tight junctions (TJs) that interconnects the endothelial cells. Combined with the absence of fenestrae and an extremely low pinocytotic activity, which inhibit transcellular passage of molecules across the barrier, these morphological peculiarities establish the physical permeability barrier of the BBB. In addition, a functional BBB is manifested by a number of permanently active transport mechanisms, specifically expressed by brain capillary endothelial cells that ensure the transport of nutrients into the CNS and exclusion of blood-borne molecules that could be detrimental to the milieu required for neural transmission. Finally, while the endothelial cells constitute the physical and metabolic barrier per se, interactions with adjacent cellular and acellular layers are prerequisites for barrier function. The fully differentiated BBB consists of a complex system comprising the highly specialized endothelial cells and their underlying basement membrane in which a large number of pericytes are embedded, perivascular antigen-presenting cells, and an ensheathment of astrocytic endfeet and associated parenchymal basement membrane. Endothelial cell morphology, biochemistry, and function thus make these brain microvascular endothelial cells unique and distinguishable from all other endothelial cells in the body. Similar to the endothelial barrier, the morphological correlate of the BCSFB is found at the level of unique apical tight junctions between the choroid plexus epithelial cells inhibiting paracellular diffusion of water-soluble molecules across this barrier. Besides its barrier function, choroid plexus epithelial cells have a secretory function and produce the CSF. The barrier and secretory function of the choroid plexus epithelial cells are maintained by the expression of numerous transport systems allowing the directed transport of ions and nutrients into the CSF and the removal of toxic agents out of the CSF. In the event of CNS pathology, barrier characteristics of the blood-CNS barriers are altered, leading to edema formation and recruitment of inflammatory cells into the CNS. In this review we will describe current knowledge on the cellular and molecular basis of the functional and dysfunctional blood-CNS barriers with focus on CNS autoimmune inflammation.

The absence of angiopoietin-2 leads to abnormal vascular maturation and persistent proliferative retinopathy

Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.

Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function.

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study

PURPOSE To evaluate the safety, tolerability and bioactivity of ascending doses of MP0112, a designed ankyrin repeat protein (DARPin) that binds with high affinity to vascular endothelial growth factor-A (VEGF-A), in treatment-naive patients with exudative age-related macular degeneration (AMD). DESIGN Phase I/II, open-label, multicenter, dose-escalation study. METHODS Patients were to receive a single intravitreal injection of MP0112 at doses ranging from 0.04 to 3.6 mg and be monitored for 16 weeks for safety, efficacy, pharmacokinetics, and dose response. RESULTS Altogether, 32 patients received a single injection of MP0112. The maximum tolerated dose was 1.0 mg because of a case of endophthalmitis in the 2.0 mg cohort. Drug-related adverse events were reported by 13 (41%) of 32 patients; they included ocular inflammation in 11 patients (7 mild, 4 moderate in severity). Visual acuity scores were stable or improved compared with baseline for ≥4 weeks following injection; both retinal thickness and fluorescein angiography leakage decreased in a dose-dependent manner. Rescue therapy was administered to 20 (91%) of 22 patients who received 0.04-0.4 mg MP0112 compared with 4 of 10 (40%) patients who received 1.0 or 2.0 mg. Of patients in the higher-dose cohorts who did not require rescue treatment, 83% (5/6) maintained reductions in central retinal thickness through week 16. CONCLUSIONS A single injection of 1.0 or 2.0 mg MP0112 resulted in mean decreases in retinal thickness and leakage area despite ocular inflammation. Larger-scale studies are warranted to confirm these observations.

β2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

Investigation of retinal morphology alterations using spectral domain optical coherence tomography in a mouse model of retinal branch and central retinal vein occlusion.

Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.

TREAT-AND-EXTEND REGIMENS WITH ANTI-VEGF AGENTS IN RETINAL DISEASES: A Literature Review and Consensus Recommendations

PURPOSE A review of treat-and-extend regimens (TERs) with intravitreal anti-vascular endothelial growth factor agents in retinal diseases. METHODS There is a lack of consensus on the definition and optimal application of TER in clinical practice. This article describes the supporting evidence and subsequent development of a generic algorithm for TER dosing with anti-vascular endothelial growth factor agents, considering factors such as criteria for extension. RESULTS A TER algorithm was developed; TER is defined as an individualized proactive dosing regimen usually initiated by monthly injections until a maximal clinical response is observed (frequently determined by optical coherence tomography), followed by increasing intervals between injections (and evaluations) depending on disease activity. The TER regimen has emerged as an effective approach to tailoring the dosing regimen and for reducing treatment burden (visits and injections) compared with fixed monthly dosing or monthly visits with optical coherence tomography-guided regimens (as-needed or pro re nata). It is also considered a suitable approach in many retinal diseases managed with intravitreal anti-vascular endothelial growth factor therapy, given that all eyes differ in the need for repeat injections. CONCLUSION It is hoped that this practical review and TER algorithm will be of benefit to health care professionals interested in the management of retinal diseases.