Brush-Like Coatings Provide a Cloak of Invisibility to Titanium Implants.

Orthopedic implants such as knee and hip implants are one of the most important types of medical devices. Currently, the surface of the most advanced implants consists of titanium or titanium-alloys with high porosity at the bone-contacting surface leading to superior mechanical properties, excellent biocompatibility, and the capability of inducing osseointegration. However, the increased surface area of porous titanium provides a nidus for bacteria colonization leading to implant-related infections, one of the main reasons for implant failure. Here, two readily applicable titanium-coatings based on hydrophilic carboxybetaine polymers that turn the surface stealth thereby preventing bacterial adhesion and colonization are developed. These coatings are biocompatible, do not affect cell functionality, exhibit great antifouling properties, and do not cause additional inflammation during the healing process. In this way, the coatings can prevent implant-related infections, while at the same time being completely innocuous to its biological environment. Thus, these coating strategies are a promising route to enhance the biocompatibility of orthopedic implants and have a high potential for clinical use, while being easy to implement in the implant manufacturing process.

Blood Milieu in Acute Myocardial Infarction Reprograms Human Macrophages for Trauma Repair.

Acute myocardial infarction (AMI) is accompanied by a systemic trauma response that impacts the whole body, including blood. This study addresses whether macrophages, key players in trauma repair, sense and respond to these changes. For this, healthy human monocyte-derived macrophages are exposed to 20% human AMI (n = 50) or control (n = 20) serum and analyzed by transcriptional and multiparameter functional screening followed by network-guided data interpretation and drug repurposing. Results are validated in an independent cohort at functional level (n = 47 AMI, n = 25 control) and in a public dataset. AMI serum exposure results in an overt AMI signature, enriched in debris cleaning, mitosis, and immune pathways. Moreover, gene networks associated with AMI and with poor clinical prognosis in AMI are identified. Network-guided drug screening on the latter unveils prostaglandin E2 (PGE2) signaling as target for clinical intervention in detrimental macrophage imprinting during AMI trauma healing. The results demonstrate pronounced context-induced macrophage reprogramming by the AMI systemic environment, to a degree decisive for patient prognosis. This offers new opportunities for targeted intervention and optimized cardiovascular disease risk management.

Accelerating the end-to-end production of cyclic phosphate monomers with modular flow chemistry.

The biocompatibility, tunable degradability and broad functionalities of polyphosphoesters and their potential for biomedical applications have stimulated a renewed interest from Chemistry, Medicinal Chemistry and Polymer Sciences. Commercial applications of polyphosphoesters as biomaterials are still hampered because of the time and resource-intensive sourcing of their corresponding monomers, in addition to the corrosive and sensitive nature of their intermediates and by-products. Here, we present a groundbreaking challenge for sourcing the corresponding cyclic phosphate monomers by a different approach. This approach relies on the use of continuous flow technologies to intensify the end-to-end preparation of cyclic phosphate monomers with a semi-continuous modular flow platform. The applied flow technology mitigates both safety and instability issues related to the more classical production of cyclic phosphate monomers. The first flow module allows safe synthesis of a library of cyclic chlorophosphite building blocks and features in-line 31P NMR real-time monitoring. After optimization on the microfluidic scale, this first module is successfully transposed toward mesofluidic scale with a daily throughput of 1.88 kg. Downstream of the first module, a second module is present, allowing the quantitative conversion of cyclic chlorophosphites with molecular oxygen toward chlorophosphate derivatives within seconds. The two modules are concatenable with a downstream semi-batch quench of intermediate chlorophosphate with alcohols, hence affording the corresponding cyclic phosphate monomers. Such a continuous flow setup provides considerable unprecedented advantages to safely and efficiently synthesize a library of versatile high value-added cyclic phosphate monomers at large scale. These freshly produced monomers can be successfully (co)polymerized, using either batch or flow protocols, into well-defined polyphosphoesters with assessed thermal properties and cytotoxicity.

(Sub)populations of extracellular vesicles released by TNF-α -triggered human endothelial cells promote vascular inflammation and monocyte migration.

Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization.

Phosphodiester Hydrogels for Cell Scaffolding and Drug Release Applications.

Given the major structural role phosphodiesters play in the organism it is surprising they have not been more widely adopted as a building block in sophisticated biomimetic hydrogels and other biomaterials. The potential benefits are substantial: phosphoester-based materials show excellent compatibility with blood, cells, and a remarkable resistance to protein adsorption that may trigger a foreign-body response. In this work, a novel class of phosphodiester-based ionic hydrogels is presented which are crosslinked via a phosphodiester moiety. The material shows good compatibility with blood, supports the growth and proliferation of tissue and presents opportunities for use as a drug release matrix as shown with fluorescent model compounds. The final gel is produced via base-induced elimination from a phosphotriester precursor, which is made by the free-radical polymerization of a phosphotriester crosslinker. This crosslinker is easily synthesized via multigram one-pot procedures out of common laboratory chemicals. Via the addition of various comonomers the properties of the final gel may be tuned leading to a wide range of novel applications for this exciting class of materials.

Extracellular Vesicles Work as a Functional Inflammatory Mediator Between Vascular Endothelial Cells and Immune Cells.

Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) in vitro. Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes.

In vitro and in vivo evaluation of drug-eluting microspheres designed for transarterial chemoembolization therapy.

Poly(D,L-lactic acid) biodegradable microspheres, loaded with the drugs cisplatin and/or sorafenib tosylate, were prepared, characterized and studied. Degradation of the microspheres, and release of cisplatin and/or sorafenib tosylate from them, were investigated in detail. Incubation of the drug-carrying microspheres in phosphate buffered saline (pH=7.4) revealed slow degradation. Nevertheless, significant release of cisplatin and sorafenib tosylate from microspheres loaded with both drugs was apparent in vitro; this can be attributed to their porous structure. Supernatants from microspheres loaded with both drugs showed strong toxic effects on cells (i.e. endothelial cells, fibroblast cells and Renca tumor cells) and potent anti-angiogenic effect in the matrigel endothelial tube assay. In vivo anti-tumor effects of the microspheres were also observed, in a Renca tumor mouse model. The poly(D,L-lactic acid) microspheres containing both cisplatin and sorafenib tosylate revealed highest therapeutic efficacy, probably demonstrating that combined local administration of cisplatin and sorafenib tosylate synergistically inhibits tumor growth in situ. In conclusion, this study demonstrates the applicability of biodegradable poly(D,L-lactic acid) microspheres loaded with cisplatin and sorafenib tosylate for local drug delivery as well as the potential of these microspheres for future use in transarterial chemoembolization.

Arginase-1 deficiency regulates arginine concentrations and NOS2-mediated NO production during endotoxemia.

RATIONALE AND OBJECTIVE: Arginase-1 is an important component of the intricate mechanism regulating arginine availability during immune responses and nitric oxide synthase (NOS) activity. In this study Arg1(fl/fl)/Tie2-Cre(tg/-) mice were developed to investigate the effect of arginase-1 related arginine depletion on NOS2- and NOS3-dependent NO production and jejunal microcirculation under resting and endotoxemic conditions, in mice lacking arginase-1 in endothelial and hematopoietic cells. METHODS AND RESULTS: Arginase-1-deficient mice as compared with control mice exhibited higher plasma arginine concentration concomitant with enhanced NO production in endothelial cells and jejunal tissue during endotoxemia. In parallel, impaired jejunal microcirculation was observed in endotoxemic conditions. Cultured bone-marrow-derived macrophages of arginase-1 deficient animals also presented a higher inflammatory response to endotoxin than control littermates. Since NOS2 competes with arginase for their common substrate arginine during endotoxemia, Nos2 deficient mice were also studied under endotoxemic conditions. As Nos2(-/-) macrophages showed an impaired inflammatory response to endotoxin compared to wild-type macrophages, NOS2 is potentially involved. A strongly reduced NO production in Arg1(fl/fl)/Tie2-Cre(tg/-) mice following infusion of the NOS2 inhibitor 1400W further implicated NOS2 in the enhanced capacity to produce NO production Arg1(fl/fl)/Tie2-Cre(tg/-) mice. CONCLUSIONS: Reduced arginase-1 activity in Arg1(fl/fl)/Tie2-Cre(tg/-) mice resulted in increased inflammatory response and NO production by NOS2, accompanied by a depressed microcirculatory flow during endotoxemia. Thus, arginase-1 deficiency facilitates a NOS2-mediated pro-inflammatory activity at the expense of NOS3-mediated endothelial relaxation.

A nontoxic additive to introduce x-ray contrast into poly(lactic acid). Implications for transient medical implants such as bioresorbable coronary vascular scaffolds.

Bioresorbable coronary vascular scaffolds are about to revolutionize the landscape of interventional cardiology. These scaffolds, consisting of a poly(L-lactic acid) interior and a poly(D,L-lactic acid) surface coating, offer a genuine alternative for metallic coronary stents. Perhaps the only remaining drawback is that monitoring during implantation is limited to two X-ray contrast points. Here, a new approach to make the biodegradable scaffolds entirely radiopaque is explored. A new contrast agent is designed and synthesized. This compound is miscible with poly(D,L-lactic acid) matrix, and nontoxic to multiple cell types. Blends of poly(D,L-lactic acid) and the contrast agent are found to be hemocompatible, noncytotoxic, and radiopaque. The data show that it is possible to manufacture fully radiopaque bioresorbable coronary vascular scaffolds. Whole-stent X-ray visibility helps interventionalists ensure that the scaffold deploys completely. This important advantage may translate into improved safety, accuracy, and clinical performance of cardiac stents.

Utilization of flax fibers for biomedical applications.

Over the past decades, a large number of animal-derived materials have been introduced for several biomedical applications. Surprisingly, the use of plant-based materials has lagged behind. To study the feasibility of plant-derived biomedical materials, we chose flax (Linum usitatissimum). Flax fibers possess excellent physical-mechanical properties, are nonbiodegradable, and there is extensive know-how on weaving/knitting of them. One area where they could be useful is as implantable mesh structures in surgery, in particular for the repair of incisional hernias of the abdominal wall. Starting with a bleached flax thread, a prototype mesh was specifically knitted for this study, and its cytocompatibility was studied in vitro and in vivo. The experimental data revealed that application of flax in surgery first requires a robust method to remove endotoxins and purify the flax fiber. Such a method was developed, and purified meshes did not cause loss of cell viability in vitro. In addition, endotoxins determined using limulus amebocyte lysate test were at acceptable levels. In vivo, the flax meshes showed only mild inflammation, comparable to commercial polypropylene meshes. This study revealed that plant-derived biomaterials can provide a new class of implantable materials that could be used as surgical meshes or for other biomedical applications.

Specific visualization of nitric oxide in the vasculature with two-photon microscopy using a copper based fluorescent probe.

To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.

Cellular decisions in cardiac outflow tract and coronary development: an act by VEGF and NOTCH.

Congenital cardiac abnormalities are, due to their relatively high frequency and severe impact on quality of life, an important focus in cardiovascular research. Recently, various human studies have revealed a high coincidence of VEGF and NOTCH polymorphisms with cardiovascular outflow tract anomalies, such as bicuspid aortic valves and Tetralogy of Fallot, next to predisposition for cardiovascular pathologies, including atherosclerosis and aortic valve calcification. This genetic association between VEGF/NOTCH mutations and congenital cardiovascular defects in humans has been supported by substantial proof from animal models, revealing interaction of both pathways in cellular processes that are crucial for cardiac development. This review focuses on the role of VEGF and NOTCH signaling and their interplay in cardiogenesis with special interest to coronary and outflow tract development. An overview of the association between congenital malformations and VEGF/NOTCH polymorphisms in humans will be discussed along with their potential mechanisms and processes as revealed by transgenic mouse models. The molecular and cellular interaction of VEGF and subsequent Notch-signaling in these processes will be highlighted.

Vascular potency of Sus scrofa bone marrow-derived mesenchymal stem cells: a progenitor source of medial but not endothelial cells.

Short-term thrombotic occlusion and compliance mismatch hamper clinical use of synthetic small-diameter tissue engineered vascular grafts. It is felt that preconditioning of the graft with intimal (endothelial) and medial (vascular smooth muscle) cells contributes to patency of the graft. Autologous, non-vessel-derived cells are preferred because of systemic vascular pathology and immunologic concerns. We tested in a porcine model whether cultured bone marrow-derived mononuclear cells, also referred to as mesenchymal stem cells (MSC), are a potential source of intimal or medial cells in vascular tissue engineering. We show that MSC cultured in endothelial medium do not gain an endothelial phenotype or functional characteristics, even after enrichment for CD31, culturing under flow, treatment with additional growth factors (vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2), or co-culture with microvascular endothelial cells (EC). On the other hand, we show that MSC cultured in MSC medium, but not in smooth muscle cell medium, show phenotypical and functional characteristics of vascular smooth muscle cells. We conclude that bone marrow-derived MSCs can be used as a bona fide source of medial, but not EC in small-diameter vascular tissue engineering.

Aberrant lymphatic development in euploid fetuses with increased nuchal translucency including Noonan syndrome.

OBJECTIVE: Increased nuchal translucency in the human fetus is associated with aneuploidy, structural malformations and several syndromes such as Noonan syndrome. In 60­70% of the Noonan syndrome cases, a gene mutation can be demonstrated. Previous research showed that aneuploid fetuses with increased nuchal translucency (NT) demonstrate an aberrant lymphatic endothelial differentiation. METHOD: Fetuses with increased NT and normal karyotype (n = 7) were compared with euploid controls having normal NT (n = 5). A Noonan syndrome gene mutation was found in three out of seven fetuses with increased NT. Endothelial differentiation was evaluated by immunohistochemistry using lymphatic markers (PROX-1, Podoplanin, LYVE-1) and blood vessel markers vascular endothelial growth factor-A (VEGF-A), Neuropilin-1 (NP-1), Sonic hedgehog, von Willebrand factor, and the smooth muscle cell marker, smooth muscle actin. RESULTS: Nuchal edema and enlarged jugular lymphatic sacs (JLSs) were observed in fetuses with increased NT, together with abnormal lymphatic endothelial differentiation i.e. the presence of blood vessel characteristics, including high levels of VEGF-A and NP-1 expression. The enlarged JLSs contained erythrocytes and were surrounded by smooth muscle cells. CONCLUSION: This study shows an aberrant lymphatic endothelial differentiation in fetuses with increased NT and a normal karyotype (including Noonan syndrome fetuses), as was previously reported before in aneuploid fetuses.

Feed-forward signaling by membrane-bound ligand receptor circuit: the case of NOTCH DELTA-like 4 ligand in endothelial cells.

The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.

Platelet-derived growth factor is involved in the differentiation of second heart field-derived cardiac structures in chicken embryos.

For the establishment of a fully functional septated heart, addition of myocardium from second heart field-derived structures is important. Platelet-derived growth factors (PDGFs) are known for their role in cardiovascular development. In this study, we aim to elucidate this role of PDGF-A, PDGF-C, and their receptor PDGFR-alpha. We analyzed the expression patterns of PDGF-A, -C, and their receptor PDGFR-alpha during avian heart development. A spatiotemporal pattern of ligands was seen with colocalization of the PDGFR-alpha. This was found in second heart field-derived myocardium as well as the proepicardial organ (PEO) and epicardium. Mechanical inhibition of epicardial outgrowth as well as chemical disturbance of PDGFR-alpha support a functional role of the ligands and the receptor in cardiac development.

Abnormal Shh and FOXC2 expression correlates with aberrant lymphatic development in human fetuses with increased nuchal translucency.

OBJECTIVE: Previous research in fetuses with increased nuchal translucency (NT) showed abnormal lymphatic endothelial differentiation characteristics, including increased vascular endothelial growth factor (VEGF)-A expression, and aberrant smooth muscle cells (SMCs) surrounding enlarged jugular lymphatic sacs (JLS). We hypothesized that abnormal Sonic hedgehog (Shh) expression would result in altered VEGF-A signaling in the lymphatic endothelial cells of the JLS and that aberrant acquisition of SMCs could be caused by downregulation of forkhead transcription factor FOXC2 and upregulation of platelet-derived growth factor (PDGF)-B in the lymphatic endothelial cells of the JLS. METHODS: Five trisomy 21 fetuses and four controls were investigated using immunohistochemistry for Shh, VEGF-A, FOXC2 and PDGF-B expression in the lymphatic endothelial cells of the JLS. RESULTS: An increased Shh, VEGF-A and PDGF-B expression, and decreased FOXC2 expression were shown in the lymphatic endothelial cells of the JLS of the trisomic fetuses. CONCLUSIONS: Increased Shh and VEGF-A expression is correlated with an aberrant lymphatic endothelial differentiation in trisomy 21 fetuses. The SMCs surrounding the JLS can possibly be explained by an increase of PDGF-B-induced SMC recruitment and/or differentiation. This underscores earlier findings that indicate the loss of lymphatic identity in trisomy 21 fetuses and a shift towards a blood vessel wall phenotype.

The alternatively spliced anti-angiogenic family of VEGF isoforms VEGFxxxb in human kidney development.

BACKGROUND/AIM: Vascular endothelial growth factor (VEGF), required for renal development, is generated by alternative splicing of 8 exons to produce two families, pro-angiogenic VEGF(xxx), formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGF(xxx)b, generated by distal splicing in exon 8 (exon 8b). VEGF(165)b, the first described exon 8b-containing isoform, antagonises VEGF(165) and is anti-angiogenic in vivo. METHODS: Using VEGF(xxx)b-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF(165)b on renal endothelial and epithelial cells. RESULTS: VEGF(xxx)b formed 45% of total VEGF protein in adult renal cortex, and VEGF(165)b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGF(xxx)b was expressed in the condensed vesicles of the metanephros, epithelial cells of the comma-shaped bodies, invading endothelial cells and epithelial cells of the S-shaped body, and in the immature podocytes. Expression reduced as the glomerulus matured. CONCLUSION: These results show that the anti-angiogenic VEGF(xxx)b isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGF(xxx)b family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGF(xxx) isoforms.