TSP-2 inhibits renal endothelial cell proliferation.

<p>Since total renal proliferation, as assessed by PCNA staining, was similar in both groups (A), TSP-2 gene therapy specifically reduced endothelial proliferative activity (B), as assessed by CD31/PCNA double positive cells (C, endothelial cells stained in brown and PCNA positive nuclei in blue). Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,007. The effect of TSP-2 on proliferation of isolated rat endothelial cells was investigated by a BrdU-incorporation assay (D, n = 6; *p<0,05).</p

Influence of TSP-2 gene therapy on VEGF/VEGF receptor expression and MMP-2 activity.

<p>VEGF and VEGF receptor 1 expression was assessed by semiquantitative scoring of immunohistochemistry. VEGF was reduced in glomeruli (A) as well as in the tubulointerstitial compartment (B) from TSP-2 treated rats. Representative pictures from VEGF stained glomeruli showing pronounced VEGF expression in podocyted as well as mesangial cells in kidneys from control plasmid treated animals (C, brown staining). In contrast, VEGF was only rarely detected in glomeruli from TSP-2 treated rats (D, brown staining). Expression of VEGF receptor 1 was similar in glomeruli (E) as well as cortex (F) of both groups. Glomerular VEGF mRNA expression was comparable in both groups (G). Serum MMP-2 activity at endpoint of the study was detected by zymography (I) and evaluated by densitometry (H). Control (n = 8) vs. TSP-2 treated (A–F:n = 8; G–I:n = 6); *p<0,05.</p

Fibronectin and alpha-smooth muscle actin staining in renal grafts treated with TSP-2 gene therapy.

<p>Representative microphotographs from immunohistological staining of kidney grafts for active TGF-β (A, control plasmid; B, TSP-2 therapy, brown cytosolic staining), P-smad 2/3 (C, control plasmid; D, TSP-2 therapy, brown nuclear staining), PAI-1 (E, control plasmid; F, TSP-2 therapy, brown staining), fibronectin (G, control plasmid; H, TSP-2 therapy, brown staining) and alpha-smooth muscle actin (I, control plasmid; J, TSP-2 therapy, brown staining) are shown.</p

Renal injury was impaired by TSP-2 gene therapy.

<p>Injury in renal transplants was assessed by semiquantitative scoring of glomerulosclerosis (A) and tubular injury (B) in PAS-stained paraffin sections as well as FSGS-lesions (C). Podocyte damage was evaluated by semiquantitative scoring of desmin positive podocytes by immunohistochemistry (D). Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,02.</p

Expression of TSP-1 and TSP-2 in F344 Lewis rat renal allograft model.

<p>Renal TSP-1 and TSP-1 expression was evaluated in biopsies from rats treated with the overexpression plasmid for luciferase control or TSP-2 using immunohistochemistry. TSP-1 expression was localized in glomeruli and within and around the Bowman's capsule (A, brown staining) as well as in fibroblasts and atrophic tubular cells (B). A semiquantitative evaluation of TSP-1 expression revealed no significant differences between the two groups (C). Staining for endogenous TSP-2 showed marked expression within some tubules (F), while glomerular staining for TSP-2 is lacking (E). Endogenous TSP-2 expression shows a tendency to increased TSP-2 expression within the TSP-2 treated group compared to the controls. Control (n = 8) vs. TSP-2 treated (n = 8).</p

Immunohistochemistry of inflammatory cells in renal grafts after TSP-2 gene therapy.

<p>Representative microphotographs from immunohistological staining of kidney grafts for CD8a positive T-cells (A, control plasmid; B, TSP-2 therapy, brown staining), MHC II positive antigen presenting cells (C, control plasmid; D, TSP-2 therapy, brown staining) and CD45R positive B-cells (E, control plasmid; F, TSP-2 therapy, brown staining) are shown.</p

TSP-2 gene therapy reduced TGF-β activation while not affecting total TGF-β expression.

<p>In gene therapy, treated rats with renal transplants TGF-β were evaluated by immunohistochemistry. Active TGF-β was either detected directly by using an antibody recognizing active TGF-β (A) or indirectly by evaluation of phosphorylation of the TGF-β signaling molecule smad2/3 (B) or expression of the TGF-β downstream target PAI-1 within the glomeruli (C) or the cortex (D). Representative microphotographs of PAI-1 staining in renal grafts treated with control (E) and TSP-2 overexpressing plasmid (F) are shown. Total TGF-β1 (G) and TGF-β2 (H) was similar in both groups. Control (n = 8) vs. TSP-2 treated (n = 8); *p<0,05.</p

Establishment of long-term therapy using pUblux vector system compared to the regular CMV promoter systems:

<p>Rat thigh muscles were transfected with either pGl2 (A–C) or pUblux (D–F) overexpression vector. Luciferase activity was measured in vivo 5 days (A, D), 4 month (B, E) and 1 year (C, F) after transfection and evaluated using ImageJ-software (G).</p

Primer sequences used for quantification of gene expression by real-time PCR.

<p>Primer sequences used for quantification of gene expression by real-time PCR.</p

Matrix deposition is not ameliorated by TSP-2 gene therapy in the chronic allograft model.

<p>Collagen IV, fibronectin and alpha-smooth muscle actin (SMA) were evaluated by semiquantitative scoring of immunohistologic staining. Collagen IV was similar in cortex (A) of both groups. Fibronectin was similar in glomeruli (B) but increased within the cortex of renal grafts from TSP-2 treated animals (C). Glomerular fibronectin mRNA shows a tendency to higher expression in TSP-2 treated animals (D) Myofibroblasts were evaluated in glomeruli (E) as well as in renal cortex (F) using alpha-smooth muscle actin (SMA) as a marker. Glomerular mRNA of SMA (G) and CTGF (H) was increased in TSP-2 treated rats compared to control. Control (n = 8) vs. TSP-2 treated (A–C, E–F:n = 8; D, G–H:n = 6); *p<0,05.</p