The mechanisms that cause tumors such as melanomas to metastasize into peripheral lymphatic capillaries are poorly defined. Non-mutually-exclusive mechanisms are lymphatic endothelial cell (LEC) ...chemotaxis and proliferation in response to tumor cells (chemotaxis-lymphangiogenesis hypothesis) or LECs may secrete chemotactic agents that attract cancer cells (chemotactic metastasis hypothesis). Using migration assays, we found evidence supporting both hypotheses. Conditioned medium (CM) from metastatic malignant melanoma (MMM) cell lines attracted LEC migration, consistent with the lymphangiogenesis hypothesis. Conversely, CM from mixed endothelial cells or LECs, but not blood endothelial cells, attracted MMM cells but not non-metastatic melanoma cells, consistent with the chemotactic metastasis hypothesis. MMM cell lines expressed CCR7 receptors for the lymphatic chemokine CCL21 and CCL21 neutralizing antibodies prevented MMM chemotaxis in vitro. To test for chemotactic metastasis in vivo tumor cells were xenotransplanted into nude mice approximately 1 cm from an injected LEC depot. Two different MMM grew directionally towards the LECs, whereas non-metastatic melanomas did not. These observations support the hypothesis that MMM cells grow towards regions of high LEC density owing to chemotactic LEC secretions, including CCL21. This chemotactic metastasis may contribute to the close association between metastasizing tumor cells and peri-tumor lymphatic density and promote lymphatic invasion.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
ABSTRACT
In attempting to quantify statistically the density structure of the interstellar medium, astronomers have considered a variety of fractal models. Here, we argue that, to properly ...characterize a fractal model, one needs to define precisely the algorithm used to generate the density field, and to specify – at least – three parameters: one parameter constrains the spatial structure of the field, one parameter constrains the density contrast between structures on different scales, and one parameter constrains the dynamic range of spatial scales over which self-similarity is expected (either due to physical considerations, or due to the limitations of the observational or numerical technique generating the input data). A realistic fractal field must also be noisy and non-periodic. We illustrate this with the exponentiated fractional Brownian motion (xfBm) algorithm, which is popular because it delivers an approximately lognormal density field, and for which the three parameters are, respectively, the power spectrum exponent, β, the exponentiating factor, ${\cal S}$, and the dynamic range, ${\cal R}$. We then explore and compare two approaches that might be used to estimate these parameters: machine learning and the established Δ-Variance procedure. We show that for 2 ≤ β ≤ 4 and $0\le {\cal S}\le 3$, a suitably trained Convolutional Neural Network is able to estimate objectively both β (with root-mean-square error $\epsilon _{_\beta }\sim 0.12$) and ${\cal S}$ (with $\epsilon _{_{\cal S}}\sim 0.29$). Δ-variance is also able to estimate β, albeit with a somewhat larger error ($\epsilon _{_\beta }\sim 0.17$) and with some human intervention, but is not able to estimate ${\cal S}$.
Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the ...pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the anti-angiogenic family formed by distal splice site selection in the terminal exon, termed VEGFxxxb, where xxx is the amino acid number. The most studied isoforms, VEGF165 and VEGF165b have been shown to be present in tumour and normal tissues respectively. VEGF165b has been shown to inhibit VEGF- and hypoxia-induced angiogenesis, and VEGF-induced cell migration and proliferation in vitro. Here we show that overexpression of VEGF165b by tumour cells inhibits the growth of prostate carcinoma, Ewing's sarcoma and renal cell carcinoma in xenografted mouse tumour models. Moreover, VEGF165b overexpression inhibited tumour cell-mediated migration and proliferation of endothelial cells. These data show that overexpression of VEGF165b can inhibit growth of multiple tumour types in vivo indicating that VEGF165b has potential as an anti-angiogenic, anti-tumour strategy in a number of different tumour types, either by control of VEGF165b expression by regulation of splicing, overexpression of VEGF165b, or therapeutic delivery of VEGF165b to tumours.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be ...painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165 a and VEGF-A165 b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165 a is upregulated and predominates over the VEGF-A165 b isoform. We show here that in rats and mice VEGF-A165 a and VEGF-A165 b have opposing effects on pain, and that blocking the proximal splicing event – leading to the preferential expression of VEGF-A165 b over VEGF165 a – prevents pain in vivo. VEGF-A165 a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165 b blocks the effect of VEGF-A165 a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxx a compared to VEGF-Axxx b, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxx a expression and reversed associated neuropathic pain. Exogenous VEGF-A165 b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxx a/VEGF-Axxx b balance by targeting alternative RNA splicing may be a new analgesic strategy.
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OBJECTIVE—Vascular endothelial growth factor (VEGF) exerts many of its effects by stimulating endothelial calcium influx, but little is known about channels mediating VEGF-induced cation entry. The ...aim of this study was to measure and characterize for the first time the VEGF-activated cation current in human microvascular endothelial cells (HMVECs).
METHODS AND RESULTS—Whole-cell patch-clamp recordings were made from HMVECs. During applied voltage ramps, VEGF activated a current that reversed at 0 mV, was sensitive to gadolinium, and required extracellular cations. Noise analysis yielded a single-channel conductance of 27 pS. The current was not dependent on intracellular calcium stores, and was not blocked by inositol triphosphate (IP3) receptor or serine/threonine kinase inhibition but was partially inhibited by flufenamic acid. A similar current was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG). To determine whether VEGF could activate recombinant ion channels with similar properties, we investigated the effect of VEGF on Chinese hamster ovary cells cotransfected with VEGFR2 and the canonical transient receptor potential (TRPC) channels, TRPC3 or TRPC6. VEGF induced a similar current to that described above in VEGFR2-TRPC3 and VEGFR2-TRPC6 cells but not in cells transfected with either cDNA alone.
CONCLUSIONS—VEGF activates a receptor-operated cation current in HMVECs and OAG can activate directly a similar current in these cells. VEGF is also able to activate heterologously expressed TRPC3/6 channels through VEGFR2.
Microvascular Research Laboratories, Department of Physiology, University of Bristol, BS2 8EJ Bristol, United Kingdom
Submitted 27 August 2003
; accepted in final form 3 October 2003
Vascular ...endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca 2+ influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca 2+ concentration (Ca 2+ i ) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity ( L p ) and fluorescence photometry to quantify changes in endothelial Ca 2+ i in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl- sn -glycerol (OAG, 100 µM), which is known to increase Ca 2+ influx through TRPCs, transiently increased L p 3.8 ± 1.2-fold (from 1.6 ± 0.8 to 9.8 ± 2.7 x 10 7 cm·s 1 ·cmH 2 O 1 ; P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 µM) did not affect the OAG-induced increases in L p . OAG also significantly increased microvascular endothelial Ca 2+ i in vivo ( n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial Ca 2+ i in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone ( P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L p in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca 2+ entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.
transient receptor potential channel; vascular endothelial growth factor; permeability; intracellular concentration; endothelium
Address for reprint requests and other correspondence: D. O. Bates, Microvascular Research Laboratories, Dept. of Physiology, Preclinical Veterinary School, Univ. of Bristol, Southwell St., Bristol BS2 8EJ, UK (E-mail: Dave.Bates{at}bristol.ac.uk ).
Malignant melanoma (MM), the most common cause of skin cancer deaths, metastasises to regional lymph nodes. In animal models of other cancers, lymphatic growth is associated with metastasis. To ...assess if lymphatic density (LD) was increased in human MM, and its association with metastasis, we measured LD inside and around archival MM samples (MM, n=21), and compared them with normal dermis (n=11), basal cell carcinoma (BCC, n=6) and Merkel cell carcinoma (MCC), a skin tumour thought to metastasise through a vascular route (MCC, n=6). Lymphatic capillary density (mm(-2)), as determined by immunohistochemical staining with the lymphatic specific marker LYVE-1, was significantly increased around MM (10.0+/-2.5 mm(-2)) compared with normal dermis (2.4+/-0.9 mm(-2)), BCC (3.0+/-0.9 mm(-2)) and MCC (2.4+/-1.4 mm(-2)) (P<0.0001). There was a small decrease in LD inside MM (1.1+/-0.7 mm(-2)) compared with normal dermis, but a highly significant decrease in BCC (0.14+/-0.13) and MCC (0.12+/-2.4) (P<0.01 Kruskal-Wallis). Astonishingly, LD discriminated between melanomas that subsequently metastasised (12.8+/-1.6 mm(-2)) and those that did not (5.4+/-1.1 mm(-2), P<0.01, Mann-Whitney). Lymphatic invasion by tumour cells was seen mainly in MM that metastasised (70% compared with 12% not metastasising, P<0.05 Fisher's Exact test). The results show that LD was increased around MMs, and that LD and tumour cell invasion of lymphatics may help to predict metastasis. To this end, a prognostic index was calculated using LD, lymphatic invasion and thickness that clearly discriminated metastatic from nonmetastatic tumours.
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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.
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Key points
Diabetes is thought to induce neuropathic pain through activation of dorsal horn sensory neurons in the spinal cord.
Here we explore the impact of hyperglycaemia on the blood supply ...supporting the spinal cord and chronic pain development.
In streptozotocin‐induced diabetic rats, neuropathic pain is accompanied by a decline in microvascular integrity in the dorsal horn. Hyperglycaemia‐induced degeneration of the endothelium in the dorsal horn was associated with a loss in vascular endothelial growth factor (VEGF)‐A165b expression. VEGF‐A165b treatment prevented diabetic neuropathic pain and degeneration of the endothelium in the spinal cord.
Using an endothelial‐specific VEGFR2 knockout transgenic mouse model, the loss of endothelial VEGFR2 signalling led to a decline in vascular integrity in the dorsal horn and the development of hyperalgesia in VEGFR2 knockout mice.
This highlights that vascular degeneration in the spinal cord could be a previously unidentified factor in the development of diabetic neuropathic pain.
Abnormalities of neurovascular interactions within the CNS of diabetic patients is associated with the onset of many neurological disease states. However, to date, the link between the neurovascular network within the spinal cord and regulation of nociception has not been investigated despite neuropathic pain being common in diabetes. We hypothesised that hyperglycaemia‐induced endothelial degeneration in the spinal cord, due to suppression of vascular endothelial growth factor (VEGF)‐A/VEGFR2 signalling, induces diabetic neuropathic pain. Nociceptive pain behaviour was investigated in a chemically induced model of type 1 diabetes (streptozotocin induced, insulin supplemented; either vehicle or VEGF‐A165b treated) and an inducible endothelial knockdown of VEGFR2 (tamoxifen induced). Diabetic animals developed mechanical allodynia and heat hyperalgesia. This was associated with a reduction in the number of blood vessels and reduction in Evans blue extravasation in the lumbar spinal cord of diabetic animals versus age‐matched controls. Endothelial markers occludin, CD31 and VE‐cadherin were downregulated in the spinal cord of the diabetic group versus controls, and there was a concurrent reduction of VEGF‐A165b expression. In diabetic animals, VEGF‐A165b treatment (biweekly i.p., 20 ng g−1) restored normal Evans blue extravasation and prevented vascular degeneration, diabetes‐induced central neuron activation and neuropathic pain. Inducible knockdown of VEGFR2 (tamoxifen treated Tie2CreERT2‐vegfr2flfl mice) led to a reduction in blood vessel network volume in the lumbar spinal cord and development of heat hyperalgesia. These findings indicate that hyperglycaemia leads to a reduction in the VEGF‐A/VEGFR2 signalling cascade, resulting in endothelial dysfunction in the spinal cord, which could be an undiscovered contributing factor to diabetic neuropathic pain.
Key points
Diabetes is thought to induce neuropathic pain through activation of dorsal horn sensory neurons in the spinal cord.
Here we explore the impact of hyperglycaemia on the blood supply supporting the spinal cord and chronic pain development.
In streptozotocin‐induced diabetic rats, neuropathic pain is accompanied by a decline in microvascular integrity in the dorsal horn. Hyperglycaemia‐induced degeneration of the endothelium in the dorsal horn was associated with a loss in vascular endothelial growth factor (VEGF)‐A165b expression. VEGF‐A165b treatment prevented diabetic neuropathic pain and degeneration of the endothelium in the spinal cord.
Using an endothelial‐specific VEGFR2 knockout transgenic mouse model, the loss of endothelial VEGFR2 signalling led to a decline in vascular integrity in the dorsal horn and the development of hyperalgesia in VEGFR2 knockout mice.
This highlights that vascular degeneration in the spinal cord could be a previously unidentified factor in the development of diabetic neuropathic pain.
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Vascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-A
xxx
and anti-angiogenic, ...anti-permeability VEGF-A
xxx
b. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-A
xxx
) and eGFP expression denotes distal splice site selection (VEGF-A
xxx
b). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-A
xxx
and VEGF-A
xxx
b. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo
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