D. O. Bates

Regulation of microvascular permeability by vascular endothelial growth factors

Generation of new blood vessels from pre‐existing vasculature (angiogenesis) is accompanied in almost all states by increased vascular permeability. This is true in physiological as well as pathological angiogenesis, but is more marked during disease states. Physiological angiogenesis occurs during tissue growth and repair in adult tissues, as well as during development. Pathological angiogenesis is seen in a wide variety of diseases, which include all the major causes of mortality in the west: heart disease, cancer, stroke, vascular disease and diabetes. Angiogenesis is regulated by vascular growth factors, particularly the vascular endothelial growth factor family of proteins (VEGF). These act on two specific receptors in the vascular system (VEGF‐R1 and 2) to stimulate new vessel growth. VEGFs also directly stimulate increased vascular permeability to water and large‐molecular‐weight proteins. We have shown that VEGFs increase vascular permeability in mesenteric microvessels by stimulation of tyrosine autophosphorylation of VEGF‐R2 on endothelial cells, and subsequent activation of phospholipase C (PLC). This in turn causes increased production of diacylglycerol (DAG) that results in influx of calcium across the plasma membrane through store‐independent cation channels. We have proposed that this influx is through DAG‐mediated TRP channels. It is not known how this results in increased vascular permeability in endothelial cells in vivo. It has been shown, however, that VEGF can stimulate formation of a variety of pathways through the endothelial cell, including transcellular gaps, vesiculovacuolar organelle formation, and fenestrations. A hypothesis is outlined that suggests that these all may be part of the same process.

Diabetic retinopathy is associated with a switch in splicing from anti- to pro-angiogenic isoforms of vascular endothelial growth factor

Aims/hypothesisProliferative diabetic retinopathy results from excess blood vessel growth into the vitreous fluid of the eye. Retinal angiogenesis is regulated by expression of vascular endothelial growth factor (VEGF), and many studies have shown that VEGF is critically involved in proliferative diabetic retinopathy. VEGF is alternatively spliced to form the angiogenic (VEGFxxx) and potentially anti-angiogenic (VEGFxxxb) family of isoforms. The VEGFxxxb family is found in normal tissues, but down-regulated in renal and prostate cancer. Previous studies on endogenous expression of VEGF in the eye have not distinguished between the two families of isoforms.MethodsWe measured VEGFxxxb isoform expression in normal human eye tissue (lens, sclera, retina and iris) and vitreous fluid using enzyme-linked immunosorbent assay and Western blotting with a VEGFxxxb-specific antibody.ResultsVEGFxxxb protein was expressed in lens, sclera, retina, iris and vitreous fluid. Multiple isoforms were seen, including VEGF165b, VEGF121b, VEGF145b, VEGF183b and VEGF189b. In non-diabetic patients, 64±7% of the VEGF in the vitreous was VEGFxxxb (n=18), whereas in diabetic patients only 12.5±3.6% of total VEGF was VEGFxxxb.Conclusions/interpretationSince VEGFxxxb inhibits VEGFxxx-induced angiogenesis in a one-to-one stoichiometric manner, these results show that in the eye of diabetic patients VEGF splicing was switched from an anti-angiogenic to a pro-angiogenic environment. This occurred through changes to the ratio of VEGFxxx : VEGFxxxb. Alterations to splicing, and through that to the balance of VEGF isoforms, could therefore be a potential therapeutic strategy for diabetic retinopathy.

Hallmarks of alternative splicing in cancer

The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial–mesenchymal and mesenchymal–epithelial transitions and metastasis. Each of the ‘hallmarks of cancer’ is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics—the alternative-splicing inhibitors.

Vascular endothelial growth factor increases Rana vascular permeability and compliance by different signalling pathways

Vascular endothelial growth factor (VEGF) chronically increases microvascular permeability, compliance and vessel diameter. To determine the signalling pathways by which VEGF exerts these effects, we investigated the role of Ca2+ influx and mitogen‐activated protein kinase (MAPK) phosphorylation on the increase in hydraulic conductivity (Lp), diameter and compliance in mesenteric microvessels in the anaesthetised frog (Rana species). The VEGF‐mediated chronically increased permeability was attenuated by co‐perfusion of VEGF with 5 mm NiCl2, previously shown to inhibit Ca2+ influx. MAPK phosphorylation inhibition by PD98059 did not affect the chronic increase in Lp. To determine whether other agonists which increased Ca2+ influx also chronically increased Lp, the effect of ATP perfusion on chronic Lp was measured. ATP perfusion also chronically increased Lp. The chronic increase in Lp was therefore dependent on an initial transient Ca2+ influx, and not MAPK activation, and was not unique to VEGF stimulation. Inhibition of Ca2+ influx did not inhibit the increase in microvascular diameter or compliance brought about by VEGF. Both these increases were inhibited by PD98059. The VEGF‐mediated increase in compliance and diameter was therefore dependent on MAPK activation, not on Ca2+ influx. The chronic increase in Lp stimulated by VEGF perfusion 24 h previously was reduced when the vessel was perfused with 5 mm NiCl2. The sustained, high Lp was therefore dependent on Ca2+ influx. The endothelial cell calcium concentration ([Ca2+]i) of vessels previously perfused with VEGF or ATP, and with a chronically increased Lp, was not significantly increased compared to [Ca2+]i of endothelial cells in vessels before agonist perfusion These experiments show that VEGF acts through different pathways to stimulate increased permeability and compliance. The data are consistent with the hypothesis that VEGF chronically increases Lp through an acute stimulation of Ca2+ influx, but increases compliance and diameter by acute stimulation of the MAPK signalling pathway. They also suggest that the increase in Lp is dependent on a sustained Ca2+ influx, even though the endothelial [Ca2+]i is not raised.

The anti‐angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo

Vascular endothelial growth factor (VEGF) is the principal agent that increases microvascular permeability during physiological and pathological angiogenesis. VEGF is differentially spliced to form two families of isoforms: VEGFxxx, and VEGFxxxb. Whereas VEGF165 stimulates angiogenesis, VEGF165b is anti‐angiogenic. To determine the effect of VEGF165b on permeability, hydraulic conductivity (Lp) was measured in individually perfused microvessels in the mesentery of frogs and rats. As with VEGF165, VEGF165b increased Lp in amphibian (2.4 ± 0.3‐fold) and mammalian (1.9 ± 0.2‐fold) mesenteric microvessels. A dose–response relationship showed that VEGF165b (EC50, 0.65 pm) was approximately 25 times more potent than VEGF165 (EC50, 16 pm) in amphibian microvessels. VEGF165 has been shown to increase permeability through VEGF receptor 2 (VEGF‐R2) signalling. However, VEGF165b increased Lp of frog vessels to the same extent in the presence of the VEGF‐R2 inhibitor ZM323881, indicating that it does not increase permeability via VEGF‐R2 signalling, and was inhibited by the VEGF receptor inhibitor SU5416 at doses that are specific for VEGF receptor 1 (VEGF‐R1). VEGF165b, in contrast to VEGF165, did not result in a sustained chronic increase in Lp. These results show that although VEGF165b is anti‐angiogenic in the mesentery, it does signal in endothelial cells in vivo resulting in a transient, but not sustained, increase in microvascular Lp, probably through VEGF‐R1.

In vivo mechanisms of vascular endothelial growth factor-mediated increased hydraulic conductivity of Rana capillaries

1 Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (Lp) in vivo. To determine the signal transduction cascade through which this is mediated, we measured the effect of inhibition of various signalling pathways on VEGF‐mediated acute increases in Lp in individually perfused frog mesenteric microvessels. 2 VEGF receptors have previously been shown to activate phospholipase C‐γ (PLCγ), protein kinase C (PKC) and MEK, the mitogen‐activated and extracellular signal‐related kinase (ERK) kinase. To determine the role of these signalling pathways we measured the effects of inhibitors of each on the VEGF‐mediated increase in Lp. 3 VEGF‐mediated increases in Lp were attenuated by pre‐treatment with the PLC inhibitor U73122, but not affected by treatment with the inactive enantiomer U73343. The PLC inhibitor was also able to attenuate the increase in Lp mediated by the inflammatory mediator ATP. 4 Inhibition of either PKC or MEK activation using the selective inhibitors bisindolylmaleimide (BIM, 1 μm) and PD98059 (30 μm), respectively, did not change the VEGF‐mediated increase in Lp. However, PD98059, BIM and U73122 all reduced phosphorylation of ERK1/2 determined by Western blot analysis with anti‐phospho‐ERK1/2 antibodies. 5 Furthermore, inhibition of the conversion of diacyl glycerol (DAG) to arachidonic acid, by perfusion with the DAG lipase inhibitor RHC80267 (50 μm), did not attenuate the increase in Lp brought about by VEGF. 6 These data suggest that VEGF acutely increases microvascular permeability in vivo through a mechanism that is dependent on PLC stimulation, but is independent of PKC or MEK activation or production of arachidonic acid from DAG. We therefore propose that VEGF acutely acts to increase Lp through the direct actions of DAG, independently of PKC or arachidonic acid.

3D Reconstruction of the Glycocalyx Structure in Mammalian Capillaries using Electron Tomography

Please cite this paper as: Arkill KP, Neal CR, Mantell JM, Michel CC, Qvortrup K, Rostgaard J, Bates DO, Knupp C, Squire JM. 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography. Microcirculation 19: 343–351, 2012.

Measurement of Hydraulic Conductivity of Single Perfused Rana Mesenteric Microvessels between Periods of Controlled Shear Stress

A new method for the determination of hydraulic conductivity in individually perfused microvessels in vivo is described. A vessel is cannulated at both ends with glass micropipettes and the fluid filtration rate across the vessel wall measured from the velocities of red cells when the pressure in the micropipettes is balanced. Hydraulic conductivity measured using this double‐cannulation method (2.6 (± 0.9) × 10−7 cm s−1 cmH2O−1) was not significantly different from that measured using a previously described technique in the same vessel (2.4 (± 0.9) × 10−7 cm s−1 cmH2O−1 using the Landis‐Michel method). Shear stress on the vessel wall was controlled by changing the difference between the inflow and outflow pressures during periods of perfusion. The volume flow through the vessel, calculated from red cell velocity either in the vessel or in the pipette, was linearly proportional to this pressure difference. Higher flow rates could only be calculated from red cell velocities in the micropipette. There was no relationship between the imposed shear stress and intervening measurements of hydraulic conductivity (r= 0.029). This novel technique has advantages over the Landis‐Michel method, which include the control of outflow resistance, the measurement of shear stress under conditions of controlled pressure, the elimination of compression damage to the vessel (since vessel occlusion is not necessary) and assessment of hydraulic conductivity over the same length of vessel throughout the experiment. The measurement of solute concentrations by indwelling micropipette electrodes and the collection of perfusate for analysis are other possibilities.

Cytosolic Ca2+ concentration and rate of increase of the cytosolic Ca2+ concentration in the regulation of vascular permeability in Rana in vivo

Vascular permeability is assumed to be regulated by the cytosolic Ca2+ concentration ([Ca2+]c) of the endothelial cells. When permeability is increased, however, the maximum [Ca2+]c appears to occur after the maximum permeability increase, suggesting that Ca2+‐dependent mechanisms other than the absolute Ca2+ concentration may regulate permeability. Here we investigate whether the rate of increase of the [Ca2+]c (d[Ca2+]c/dt) may more closely approximate the time course of the permeability increase. Hydraulic conductivity (Lp) and endothelial [Ca2+]c were measured in single perfused frog mesenteric microvessels in vivo. The relationships between the time courses of the increased Lp, [Ca2+]c and d[Ca2+]c/dt were examined. Lp peaked significantly earlier than [Ca2+]c in all drug treatments examined (Ca2+ store release, store‐mediated Ca2+ influx, and store‐independent Ca2+ influx). When Lp was increased in a store‐dependent manner the time taken for Lp to peak (3.6 ± 0.9 min during store release, 1.2 ± 0.3 min during store‐mediated Ca2+ influx) was significantly less than the time taken for [Ca2+]c to peak (9.2 ± 2.8 min during store release, 2.1 ± 0.7 min during store‐mediated influx), but very similar to that for the peak d[Ca2+]c/dt to occur (4.3 ± 2.0 min during store release, 1.1 ± 0.5 min during Ca2+ influx). Additionally, when the increase was independent of intracellular Ca2+ stores, Lp (0.38 ± 0.03 min) and d[Ca2+]c/dt (0.30 ± 0.1 min) both peaked significantly before the [Ca2+]c (1.05 ± 0.31 min). These data suggest that the regulation of vascular permeability by endothelial cell Ca2+ may be regulated by the rate of change of the [Ca2+]c rather than the global [Ca2+].

Abstract 2749: SRPK1 inhibition and modulation of VEGF alternative splicing as a potential therapeutic strategy in prostate cancer

Angiogenesis is required for tumor growth and is induced principally by VEGF-A. VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer. In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by SRPK1. Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared to benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in prostate cancer may be under control of SRPK1 activity. SRPK1 levels were up-regulated in several prostate cancer cell lines in comparison with normal prostate epithelial cells. A switch in the expression of VEGF165 towards the anti-angiogenic isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knock-down (KD). PC-3 -SRPK1 KD cells resulted in tumors that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1 KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Moreover, VEGF over-expression from a splicing-insensitive construct in PC-3/SRPK1 KD cells rescued tumor growth. Small molecule inhibitors of SRPK1 are able to switch splicing towards the anti-angiogenic isoform, VEGF165b in PC3 cells in vitro and decrease tumor growth when administered intraperitoneally in vivo in a mouse model of orthotopic prostate cancer. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumor angiogenesis by regulation of VEGF splicing can alter prostate tumor growth and supports further studies into the use of SRPK1 inhibition as a potential anti-angiogenic therapy in prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Athina Mavrou, Dave Bates, Sebastian Oltean. SRPK1 inhibition and modulation of VEGF alternative splicing as a potential therapeutic strategy in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2749. doi:10.1158/1538-7445.AM2014-2749