Revealing the role of phospholipase Cβ3 in the regulation of VEGF-induced vascular permeability

Abstract

VEGF induces vascular permeability (VP) in ischemic diseases IntroductionVascular permeability (VP) plays an integral role in the pathology of cardiovascular disease, stroke, and cancer. VEGF was originally discovered as "vascular permeability factor" described as being a tumor-secreted factor that promotes microvascular permeability potently. 1 It was later discovered separately as VEGF, an endothelial mitogen 2 essential for the development of blood vessels. [3][4][5] Ischemia resulting from cardiac infarction or stroke promotes VEGF expression, which leads to hyperpermeability, edema, and tissue damage. 6,7 Angiogenesis is a key restorative mechanism in response to ischemia, 8,9 creating the therapeutical challenge of regulating the negative and beneficial actions of VEGF temporally to reduce edema and improve ischemic tissue. In cancer, VEGFmediated VP promotes tumor-cell extravasation through damaged endothelial cell junctions, often leading to widespread metastasis. 10 In addition, VEGF expression induces tumor angiogenesis through extravasation of plasma proteins into the surrounding tissue to develop a provisional matrix capable of supporting vascular sprouting and tumor growth. 1 Despite the prevalent role of VEGF-mediated VP in the pathophysiology of cardiac and cerebral diseases and cancer, the genetic regulation of VEGF-induced VP remains unclear because of the lack of an adequate, highthroughput in vivo model with which to assess quantitatively the cumulative contributions of receptors and transduction molecules to VEGF-mediated VP.New methods are necessary to elucidate genetic regulators of VP. Present in vivo VP models, such as the Miles assay, 11,12 the dual-isotopemodified Miles assay, 13 the in vivo peripheral permeability assay, 14 and intravital microscopy, 15-17 have various disadvantages, including the requirement for expensive and time-intensive murine knockout models for genetic studies. The zebrafish (Danio rerio) is a vertebrate with an optically clear embryo that allows high-resolution live imaging and is amenable to high-throughput genetic manipulation, but has yet to be used as an in vivo model to study VEGF-induced VP. In the present study, we undertook the development of a heat shock-inducible zebrafish VEGF model through which VP can be visualized and quantitated in real-time using microangiography of fluorophoreconjugated dextrans. Protein translation-blocking morpholinos (MO) microinjected into VEGF-inducible zebrafish embryos represent a relatively inexpensive and high-throughput means of identifying regulators of VEGF-mediated VP. We demonstrate the utility of this newly developed approach by identifying phospholipase C␤3 (PLC␤3) as a regulator of VEGF-mediated VP.VEGF mediates its activities through 2 receptor tyrosine kinases, VEGFR2 and VEGFR1, [18][19][20] with neuropilin acting as a coreceptor. [21][22][23] Downstream signaling events induced by VEGF include the serine phosphorylation of PLC␤3 24 and tyrosine phosphorylation of PI3K and PLC␥. 25 PLC isoforms mediate the h...

Presynaptic Inhibition Selectively Gates Auditory Transmission to the Brainstem Startle Circuit

“…The hsp:B3 recombinase transgenic line ( Tg(myl7:GFP-hsp70l:B3r-2a-Cer)y560 ) uses the heat shock protein 70l promoter (plus exon 1 and intron 1) (kind gift of Karl Clark, Mayo) [49] to drive B3 recombinase (B3r) from Zygosaccharomyces bisporus [16], followed by a 2a-peptide and Cerulean fluorescent protein. B3r and the 2a-Cer cassette were codon optimized for zebrafish [48] (Genscript).…”

Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier

“…This is the first evidence, to our knowledge, that a PLC isoform can have a barrier protective effect in the endothelium. This runs contrary to the known effects of other members of this family, such as PLCβ3, which have been reported to increase endothelial permeability[33]. We believe this unique capability is due to the presence of the CDC25 Rap GEF domain.…”

RhoC maintains vascular homeostasis by regulating VEGF-induced signaling in endothelial cells

“…We have previously described a heat-inducible VEGF zebrafish model used in conjunction with MO-mediated protein knockdown that can assess genetic regulation of vascular permeability in real time (Hoeppner et al, 2012). In Danio rerio, five Rho gene isoforms exist, Rhoaa, Rhoab, Rhoac, Rhoad and Rhoae, and the protein similarity of all human and zebrafish Rho subfamily members is 79.1% with 97.4% overall similarity (Salas-Vidal et al, 2005).…”

“…One-cell stage transgenic VEGF-inducible zebrafish (Hoeppner et al, 2012) embryos were arrayed in an agarose microinjection template and 1.5 nl of Cre mRNA (12.5 ng/μl) was microinjected into the cell of the embryo. Rhoad (5′-TCCACCTGCAGATCATAATTGGGTA-3′) and Rhoae (5′-TCCACCTGCAGATCATAATTGGGTA-3′) MOs were designed, purchased from Gene Tools, LLC (Philomath, OR), and microinjected into embryos at the one-or two-cell stage.…”