Expression of protein kinase CK2 in astroglial cells of normal and neovascularized retina

Abstract

We previously documented protein kinase CK2 involvement in retinal neovascularization. Here we describe retinal CK2 expression and combined effects of CK2 inhibitors with the somatostatin analog octreotide in a mouse model of oxygen-induced retinopathy (OIR). CK2 expression in human and rodent retinas with and without retinopathy and in astrocytic and endothelial cultures was examined by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction. A combination of CK2 inhibitors, emodin or 4,5,6,7-tetrabromobenzotriazole, with octreotide was injected intraperitoneally from postnatal (P) day P11 to P17 to block mouse OIR. All CK2 subunits (alpha, alpha', beta) were expressed in retina, and a novel CK2alpha splice variant was detected by reverse transcriptase-polymerase chain reaction. CK2 antibodies primarily reacted with retinal astrocytes, and staining was increased around new intraretinal vessels in mouse OIR and rat retinopathy of prematurity, whereas preretinal vessels were negative. Cultured astrocytes showed increased perinuclear CK2 staining compared to endothelial cells. In the OIR model, CK2 mRNA expression increased modestly on P13 but not on P17. Octreotide combined with emodin or 4,5,6,7-tetrabromobenzotriazole blocked mouse retinal neovascularization more efficiently than either compound alone. Based on its retinal localization, CK2 may be considered a new immunohistochemical astrocytic marker, and combination of CK2 inhibitors and octreotide may be a promising future treatment for proliferative retinopathies.

Figure 1

CK2α/α′ subunits are predominantly expressed in retinal astrocytes. Top row: Retina from a nondiabetic patient; double-immunohistochemical staining. CK2α/α′ completely co-distributes in the nerve fiber layer of the inner retina with retinal astrocyte marker, GFAP (right, double staining). Second row: Retina from a nondiabetic patient. Anti-CK2α/α′ mAb 1AD9 labels glial cell projections that closely appose blood vessels. No co-distribution with Müller cell marker, vimentin, is seen with double staining (right). Third row: Retina from a nondiabetic patient. On oblique section, it is clearly seen that CK2-positive astrocytes wrap around a blood vessel; Müller cell processes positive for vimentin are more distant from the vessel (right, double staining). Bottom row: Retina from a patient with DR. Müller cell processes marked by vimentin immunoreact with anti-GFAP antibody as well, indicating up-regulation of GFAP in reactive glial cells. However, CK2α/α′ does not highlight Müller cells and is revealed only in astrocytes. Asterisks mark blood vessels.

Figure 2

Specificity of immunoreactivity of human retinal astrocytes with mAb 1AD9. Top row: A dramatic decrease of astrocyte immunostaining after depletion of anti-CK2α/α′ antibody 1AD9 by incubation with a peptide comprising the specific epitope. Bottom row: Complete co-distribution of retinal CK2 staining using mAb 1AD9 and a polyclonal antibody H-286 used for rodent studies.

Figure 3

Human retinal astrocytes express α, α′, and β subunits of CK2. Only astroglial cells are stained by polyclonal rabbit antibodies to subunit-specific peptides. Each of these antibodies [anti-α (top row), anti-α′ (middle row), and anti-β (bottom row)] co-distribute with 1AD9 mAb (CK2α/α′) used here as an astrocyte marker (left). Some nonspecific background staining by polyclonal antibodies can be seen in the inner limiting membrane (ILM) and occasionally, in the cell nuclei of the inner nuclear layer. Normal human retina is shown. Right: Triple label images, DAPI staining shows nuclei.

Figure 4

CK2 immunostaining of human astrocyte and endothelial cultures. A–C: Embryonic brain astrocytes HAST040. A: Perinuclear areas stain strongly for CK2. B: Same structures stain for GFAP. C: Significant co-distribution of CK2 and GFAP revealed by color overlap (yellow). D and E: Staining of adult astrocytes from brain (D, NHA) and optic nerve head (E, ONHA) reveals similar albeit less intense staining for CK2. F: Brain microvascular endothelial cells (HBMVECs) display diffuse cytoplasmic staining for CK2. In all plates, nuclei are counterstained with DAPI (blue color).

Figure 5

RT-PCR analysis of CK2 subunit expression in normal human retina. A: Lane 1, CK2α 3′-end, 151 bp (variants 1, 2, 3); lane 2, CKα exons 1 to 4, 562, 446, 245 bp (variants 1, 2, 3, respectively), the 245-bp band (variant 3) is barely visible here; lane 3, CKα variant 2, 214 bp (primers in the boundaries of exons 1/3 and 3/4); lane 4, CKα variant 3, 191 bp; absent here (primers in the boundaries of exons 1 and 1/4); lane 5, CK2α variant 1, 225 bp (primers in exons 2 to 3); lane 6, α′ 3′-end, 163 bp; lane 7, β 3′-end, 158 bp; M, 100-bp DNA ladder. B: A 40-cycle RT-PCR of CK2α with primers in the 3′ end (as in A, lane 1). Bands corresponding to all three variants (1 at 562 bp, 2 at 446 bp, and 3 at 245 bp) are seen. Variant 2 is the major one in the retina. C: Schematic of CK2α splice variants. Variant 3 gives rise to a shorter form because the initial translation start site is spliced out.

Figure 6

CK2α/α′ expression is increased in the mouse OIR model. Control retina (top row): Anti-CK2α/α′ antibody labels astrocytes as does GFAP. OIR model (second and third rows; same section): CK2 staining is increased in retinopathy, in areas of neovascularization. CK2 staining primarily co-distributes with GFAP (second row, right; yellow color). In neovascularized areas, CK2-positive astrocytes ensheathe new blood vessels (third row; section was photobleached and then restained for basement membrane protein, perlecan, to highlight viable blood vessels, arrows). Emodin-treated retina (bottom): after treatment, CK2- and GFAP-staining patterns are similar to normal. DAPI staining shows nuclei. Right: Triple label images.

Figure 7

CK2α/α′ is not expressed around preretinal neovessels in the mouse OIR model. Top row: Strong staining of astrocytes for CK2α/α′ and GFAP. Both proteins co-distribute in astrocytes (right, yellow color) but GFAP is also seen in Müller cell transverse projections (right, green color). Nuclei of glial marker-negative preretinal vessels stained with DAPI are marked with arrows. Bottom row: Astrocytes positive for CK2 and GFAP (left and middle) only surround intraretinal but not preretinal (arrows) vessels. Preretinal vessels are positive for mural cell marker, desmin (right). Triple label immunohistochemistry.

Figure 8

Quantitative analysis of CK2 expression in the OIR model. A: QPCR reveals ∼20% increase of message levels of all three CK2 subunits in the OIR group (O) compared to normoxic controls (N) at day P13. At day P17, CK2 levels in OIR group dropped to ∼80% of normoxic controls group. Bars, mean ± SD (three experiments). B: Western blot analysis of retinal extracts for CK2α from the same samples as in A. Left: Lack of change in CK2α levels in OIR (O) versus normoxic (N) retinas compared to β-tubulin at days P13 and P17. 1× and 1.5× loading amounts for each sample are presented to show the reproducibility and sensitivity. Right: GFAP amount is increased approximately twofold in the OIR (O) versus normoxic (N) retinas compared to β-tubulin at days P13 and P17.

Figure 9

Combination of emodin or TBB with octreotide more efficiently reduces mouse retinal neovascularization than any single drug alone. Counts of preretinal nuclei as a measure of neovascularization in various groups of mice are shown. Intraperitoneal treatment with 30 mg/kg/day of emodin reduced retinal neovascularization by ∼57%, and with TBB, by 46%. Treatment with 5 mg/kg/day of octreotide yielded ∼67% reduction, and with 1 mg/kg/day of octreotide, ∼50% reduction. Emodin combined with 1 mg/kg/day octreotide reduced neovascularization by 69%, and TBB combined with 1 mg/kg/day octreotide, by 61%. Ten sections per eye from each mouse were counted. Five mice were used per each group in three independent experiments. Vehicle represents emodin solvent because octreotide solvent was just PBS. Bars, mean ± SD. *P < 0.001 versus vehicle, **P < 0.05 versus single drug or vehicle.