PLCβ2 Promotes VEGF-Induced Vascular Permeability

Abstract

Background: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need.

Methods: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) β2.

Results: Global knock-out of PLCβ2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCβ2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCβ2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCβ2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls.

Conclusions: The results implicate PLCβ2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCβ2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

Keywords: homeostasis; ischemia; permeability; reperfusion; vascular endothelial growth factor A.

Figure 1 -. VEGFA-induced dermal vascular permeability is reduced by loss of PLCβ2. Wild-Type, PLCβ2-null and PLCβ3-null

animals were injected IV with FITC-dextran then exposed locally to VEGFA (closed circles) or PBS control (open squares) via intradermal injection into the ears. Animals were imaged over 45 mins for FITC-dextran extravasation and quantified. A. Wild Type. (n = 7) B. PLCβ2-null. (n = 7) C. PLCβ3-null. (n=8).

Figure 2 -. PLCβ2-null MLEC display a reduced VEGFA-induced permeability and calcium release in vitro.

Primary lung endothelial cells were isolated from wild type, PLCβ2-null and PLCβ3-null animals and evaluated for endothelial permeability in a transwell assay. A. MLECs were treated with PBS control (NT), VEGFA or histamine and quantified for FITC in the lower chamber. (n = 3/treatment) Representative of at least three separate cell isolations and assays. B. Wild type (green) and PLCβ2-null (red) MLECs were treated with PBS control or VEGFA and intracellular calcium release was quantified (n = 10–15 individual cells/condition). Arrowhead indicates time of VEGFA treatment.

Figure 3 -. TIME shPLCβ2 cells display reduced total calcium flux after treatment with VEGFA.

TIME control and PLCβ modified cells were established with shGFP, shPLCβ2 and shPLCβ3 lentiviral constructs and antibiotic selection. A. Target specific knock-down was verified via qRT-PCR for PLCβ2 and PLCβ3 mRNA levels. B-C. shGFP (green), shPLCβ2 (red) and shPLCβ3 (blue) TIME cells were assayed in a 96-well calcium flux assay following treatment with VEGFA or histamine. D. shGFP (green), shPLCβ2 (red) and shPLCβ3 (blue) TIME cells were evaluated in an intracellular calcium release (no extracellular calcium) and extracellular calcium entry assay (extracellular calcium). (n ≥ 24 individual cells/condition). Arrowhead indicates time of VEGFA/histamine treatment.

Figure 4 -. Basal and VEGFA stimulated PIP2 levels are reduced in TIME cells with modulated PLCβ2 expression.

A. Basal PIP2 levels were quantified in shGFP, shPLCβ2 (shβ2) and shPLCβ3 (shβ3). (n = 3/condition) B. PIP2 levels following treatment with VEGFA were quantified at time 0-, 15- and 30-mins post treatment in shGFP, shPLCβ2 (shβ2) and shPLCβ3 (shβ3) TIME cells. (n = 3/condition)

Figure 5 -. PLCβ2-null animals are resistant to hyperoxia-induced lung damage.

Wild-Type and PLCβ2-null animals were exposed to hyperoxic conditions for 72hrs then moved to normoxic (room air) conditions for 4hrs. A. Average fluorescence detected in BAL following IV FITC-Dextran dosing and lung lavage. B. Body weight (g) (change from time 0) and tissue histology end points (mean cord length and peri-arteriole edema) were quantified. Representative H&E images of WT and PLCβ2-null tissues are shown. Bar = 100uM. Dotted lines show areas of edema. (n = 4/group)

Figure 6 -. PLCβ2-null animals are protected from cardiac ischemia:reperfusion damage.

Wild-Type and PLCβ2-null animals were evaluated in a cardiac ischemia:reperfusion model of LAD ligation. A. Representative images gross tissue permeability of wild type (WT) control (Sham) or treated (I:R). Asterix indicates the site of LAD. Carrot highlights areas of increased permeability. B. Representative images of PLCβ2-null control (Sham) or treated (I:R) following 30mins of I:R. Asterix indicates the site of LAD. Carrot highlights areas of increased permeability. Bar = 1 mm. C. Representative images from Wild-Type and PLCβ2-null trichrome stained tissue sections from the apex (1) to the base (6) of the heart. D. Quantification of heathy tissue (red) and infarct region (blue) normalized to percent total tissue. (n = 4–6/group)