Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

<div><p>Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.</p></div

Characterization of phospholipase C ε, ζ and η isoforms in native arteries.

<p><b>A.</b> The presence of mRNA for phospholipase C (PLC) ε, ζ, η1 and η2 isoforms was determined in mesenteric arteries (MA), pulmonary arteries (PA) and middle cerebral arteries (MCA) by PCR. Typical agarose gel electrophoresis of the PCR products showed the expression profile of PLCε, ζ, η1 and η2 isoforms in the different vascular beds and brain or testis were used as control tissue. n = 3. <b>B.</b> Quantitative real time PCR analysis of mRNA expression levels of PLCε, ζ, η1 and η2 isoforms in MA and freshly isolated endothelial cells (ECs) from MA, PA and MCA. Bar graphs show the expression of PLCε (a), ζ (b), η1 (c) and η2 (d) isoforms in MAECs, PAECs, MCAECs, MA and testis as positive control for ζ. n = 3. * P<0.05 between MAECs and MCAECs; # P<0.05 between PAECs and MCAECs; † P<0.05 between MCAECs and MA.</p

Characterization of phospholipase C β isoforms in native arteries.

<p><b>A.</b> The presence of mRNA for phospholipase C (PLC) β isoforms was determined in mesenteric arteries (MA), pulmonary arteries (PA) and middle cerebral arteries (MCA) by PCR. Typical agarose gel electrophoresis of the PCR products showed the expression profile in different vascular beds. Brain and blood were used as positive control tissues. n = 3. <b>B.</b> Quantitative real time PCR analysis of mRNA expression levels of PLCβ isoforms in MA and freshly isolated endothelial cells (ECs) from MA, PA and MCA. Bar graphs show the expression profile of PLCβ1 (a), β2 (b), β3 (c) and β4 (d) isoforms in MAECs, PAECs, MCAECs, MA and blood as control for β2. n = 3. * P<0.05 between MAECs and MCAECs; # P<0.05 between PAECs and MCAECs; † P<0.05 between MCAECs and MAs; ‡ P<0.05 between control tissue and MA. <b>C.</b> (a) Representative immunoblots of murine MA and brain that were obtained using the primary antibody anti-PLC β3 (Abcam #ab52199). GAPDH was used as reference protein. Relevant molecular weight markers are indicated on the left. n = 3. (b) Intracellular distribution of PLCβ3 immunoreactivity in ECs. (Left) Typical image showing labelling of PLC β3 in red and nuclei in blue; scale = 10 μm. (Right) Labelling of PLC β3 (red) overlay with internal elastic lamina (IEL; green) where voids correspond to potential myoendothelial projections; nucleus in blue; scale = 10 μm; n = 4.</p

Characterization of phospholipase C γ isoforms in native arteries.

<p><b>A.</b> The presence or mRNA for phospholipase C (PLC) γ isoforms was determined in mesenteric arteries (MA), pulmonary arteries (PA) and middle cerebral arteries (MCA) by PCR. Typical agarose gel electrophoresis of the PCR products showed the expression profile in the different vascular beds and brain was used as control tissue. n = 3. <b>B.</b> Quantitative real time PCR analysis of mRNA expression levels of PLCγ isoforms in MA and freshly isolated endothelial cells (ECs) from MA, PA and MCA. Bar graphs show the expression profile of PLCγ1 (a) and γ2 (b) isoforms in MAECs, PAECs, MCAECs and MA. n = 3. <b>C.</b> (a) Representative immunoblots of murine MA and brain that were analyzed using the primary antibody anti-PLC γ2 (Abcam #ab18983). GAPDH was used as reference protein. Relevant molecular weight markers are indicated on the left. n = 3. (b) Intracellular distribution of PLCγ2 immunoreactivity in ECs. (Left) Typical image showing labelling of PLCγ2 in red and nuclei in blue; scale = 10 μm. (Right) Labelling of PLCγ2 (red) overlay with internal elastic lamina (IEL; green) where voids correspond to potential myoendothelial projections; nucleus in blue; scale = 10 μm; n = 4.</p

Summary of mRNA expression for phospholipase C isoforms.

<p><i>MAECs</i>, <i>mesenteric arteries endothelial cells; PAECs</i>, <i>pulmonary arteries endothelial cells; MCAECs</i>, <i>middle cerebral arteries endothelial cells; MA</i>, <i>mesenteric arteries; PA</i>, <i>pulmonary arteries; MCA</i>, <i>middle cerebral arteries</i>.</p><p>Summary of mRNA expression for phospholipase C isoforms.</p