Inhibition of TGF-β decreases retinal perfusion and vascular autoregulation.

<p>After fourteen days of Ad-sEng and Ad-null expression, mice were injected with h.m.w. FITC-dextran through the left ventricle to localize perfused vessels. (A) Confocal analysis of retinal flat-mounts revealed reduced perfusion of the retina in the sEng-expressing mice compared to the control (representative photo of n = 12 mice). Scale bar = 200 µm. (B) The perfused vessels were visualized on retinal flat mounts by comparing the co-localization of type IV collagen (Cy3-red) and FITC and quantified on cryosections by comparing the number of vessels in the innermost vascular plexus (arrowheads) positive for both type IV collagen- and FITC to the number of vessels positive for type IV collagen but negative for FITC (C). (D) The retinas of sEng expressing mice show a marked reduction in the number of perfused vessels (n = 5, ** p<0.01). (E) After seven days of adenoviral expression, blood flow rates in the tail were measured non-invasively in response to intravascular injection of ACh. Measurements were made over 5 cycles pre-injection of ACh, normalized to 1 for each animal, and averaged at 5-cycle intervals post ACh injection. In Ad-null control mice, ACh increased tail vein blood flow rates 6–10 cycles post-injection, whereas blood flow rates were unchanged in Ad-sEng expressing mice (Ad-null: 1.619 µl/cycle, n = 5; Ad-sEng: 0.960 µl/cycle, n = 4, * p<0.01). Injection of 100 µl of saline in Ad-null or Ad-sEng mice elicited no response.</p

Inhibition of TGF-β decreases endothelial barrier function in vitro.

<p>(A) SEM analysis demonstrated a characteristic endothelial cobblestone morphology in EC monocultures. Co-culture enhanced the association between adjacent cells, which was reversed with addition of SB-431542. Scale bars = 100 µm (left panel); 10 µm (right panel). Cell size was calculated by tracing individual ECs using ImageJ. Morphology of 10T1/2 cells from cocultures with ECs was not visibly altered when compared to 10T1/2 monoculture or with the addition of SB431542. (B) Analysis of EC size in monoculture or coculture with 10T1/2 cells (+/−SB431542). Coculture did not significantly alter average cell size, however, EC size were more uniform when compared to EC mono-cultures. Addition of SB431542 led to larger and more heterogenous cell size.</p

Inhibition of TGF-β impairs endothelial tight junction protein association.

<p>(A) Assay of tight junction components in Transwell cocultures of EC and 10T1/2 cells. Immunoprecipitation of occludin and western blotting for zo-1 revealed a significant increase in association between occludin and zo-1 in coculture compared to monocultures (* p<0.05), which was reversed by the addition of SB-431542. Non-specific IgG was used as a control for IP. (B) Assay of permeability in Transwell cocultures. Coculture of BAECs with 10T1/2 cells increased EC barrier function as measured by decreased flux of FITC-dextran from the upper to lower chamber. Addition of SB-431542 increased permeability of BAEC/10T1/2 cocultures.</p

Inhibition of TGF-β increases retinal cell apoptosis.

<p>(A) Following 14 days of infection with Ad-null or Ad-sEng, retinas were examined for apoptotic cells via TUNEL staining. Retinas of sEng-expressing mice displayed a significant increase in the number of apoptotic cells. In the control retinas, occasional cells of the inner nuclear layer (INL) could be marked as apoptotic (arrowheads). Positive control tissue sections were treated with DNAse enzyme. Retinal sections were lightly counter-stained with hematoxylin QS to reveal nuclei (blue). Expression of sEng led to the induction of apoptosis of most of the cells in both the ganglion cell layer (arrows) and in the INL (arrowheads). Scale = 100 µm. (B) Lysates of whole retinas were analyzed via western blotting for cleaved caspase 3 levels. Inhibition of TGF-β led to increased cleaved caspase 3 as compared to control mice. (C) Quantification of (B) (* p<0.05).</p

Co-culture of endothelial/mesenchymal cells activates endothelial smad2/3 signaling.

<p>(A) Transient transfection of ECs with CAGA-Luc smad2/3 reporter revealed that co-culture with 10T1/2 cells activated smad2/3 signaling. Pharmacological inhibition of TGF-β with 10 µM SB-431542 reversed this activation. (B) Co-culture of ECs with 10T1/2 cells did not alter phosphorylation of EC smad1/5/8.</p

Systemic inhibition of TGF-β decreases retinal smad2 phosphorylation <i>in vivo</i>.

<p>(A) RT-PCR of adult murine retinal cDNA revealed the presence of smad2, smad3, TGF-β1 and TGF-β3. Lanes one and two are two retinal cDNA preparations. Lane three is a negative control with no RT enzyme. Immunohistochemical localization of pp-smad2 in retinas from (B) control (non infected animals) and (C) Ad-null and Ad-sEng-expressing mice (day 7) revealed pp-smad2 staining in nuclei of the ganglion cell layer (arrows) and the INL (arrowheads). sEng expression led to a decrease in the intensity of pp-smad2 staining. Scale = 50 µm. (D) Higher magnification of ppsmad2 staining in control mice, demonstrating nuclear pp-smad2 in retinal ECs (arrow). Scale = 20 µm. (E) Western blot analysis of pp-smad2, smad2, pp-smad1/5/8 and GAPDH in mouse retinas from Ad-null and Ad-sEng-expressing mice (day 7). sEng-expressing mice displayed a decrease in retinal pp-smad2 (F). Each lane corresponds to a protein preparation from one mouse retina. Quantification of ppsmad2 (n = 4, * p<0.05).</p

Inhibition of TGF-β signaling in EC-10T1/2 cell coculture leads to increased EC apoptosis.

<p>(A–B) Transwell cocultures of BAECs and 10T1/2 cells in the presence and absence of SB-431542, an inhibitor of ALK5. (A) Coculture of BAEC and 10T1/2 did not alter baseline levels of BAEC apoptosis. Addition of SB-431542 led to a significant increase in BAEC apoptosis in cocultures, but not monocultures. (B) Culture of 10T1/2 cells in the presence of BAECs significantly decreased baseline rates of 10T1/2 cell apoptosis. Addition of SB-431542 did not alter 10T1/2 cell apoptosis in either monoculture or in coculture with BAECs. Addition of SB-431542 increased cleavage of EC caspase 3 in co-culture with 10T1/2 cells (C) whereas the anti-apoptotic BCl-xl decreased (D). (E) Coculture of BAECs (unlabelled) in direct coculture with 10T1/2 cells (red) led to the formation of tube-like structures by EC, with 10T1/2 cells wrapped around and in close association with tubes. Addition of SB-431542 to BAEC-10T1/2 cocultures, after tubes had formed, led to the dissociation of 10T1/2 cells from the ECs and disassembly of tube-like structures. (F) Annexin V FACs assay for apoptosis of BAECs and 10T1/2 cells from (E) demonstrated a significant increase in EC apoptosis. Cells were retrieved using Matrigel dissociation solution and distinguished as red (10T1/2) or unlabeled (BAEC). Baseline apoptotic rates of both cell types in Matrigel are high as the cells that do not form tubes remain in the matrix and undergo apoptosis. Each experiment is representative of at least three independent experiments with similar results.</p

Inhibition of TGF-β leads to functional and morphological changes in retinal neural cells.

<p>(A) Following seven days of infection with Ad-null or Ad-sEng, retinal function was examined by scotopic ERG recording. (B) Inhibition of TGF-β did not alter the a-wave response, however b-wave was significantly decreased in Ad-sEng mice (n = 5, * p<0.05). (C) TEM of retinas from mice expressing Ad-null or Ad-sEng (day 14). Ganglion cells of sEng-expressing mice displayed features characteristic of apoptosis, with condensed nuclei (white arrow), cellular shrinkage and separation from surrounding extracellular matrix. Microvessels in Ad-null-infected mice appear normal in GCL and in the INL. Apoptosis was evident in the INL of sEng-expressing mice with separation of cells from surrounding tissue creating empty spaces (arrows) and apoptosis was apparent in the ONL of some sEng-expressing mice. Scale = 5 µm.</p

Ultrastructure of retinal vasculature following inhibition of TGF-β.

<p>(A) TEM micrograph of a microvessel in the ganglion cell layer from a retina of a control mouse expressing Ad-null (day 14). Nuclei of an EC and pericyte are apparent, with a defined basement membrane (arrow). (B)–(F) TEM micrographs of the retinal vessels from mice expressing Ad-sEng. (B) The lining of some ECs appeared ‘ruffled’ with finger-like processes protruding into the luminal space and multiple vacuoles within the cytoplasmic space (arrow). Nuclear condensation characteristic of apoptosis was apparent in some (C) pericytes and (D) ECs (arrows). (E) (F) Numerous vessels in the inner retinal layers displayed significant reductions in luminal diameter (arrows). (A)–(F) Scale = 2 µm.</p