Helmut Hopfer

Canstatin, a Novel Matrix-derived Inhibitor of Angiogenesis and Tumor Growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the α2 chain of type IV collagen, was produced as a recombinant molecule inEscherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.

Reducing Immunosuppression Preserves Allograft Function in Presumptive and Definitive Polyomavirus‐Associated Nephropathy

Early detection of polyomavirus BK (BKV) viremia and reduction of immunosuppression is recommended for preventing polyomavirus‐associated nephropathy (PyVAN), but systematic histological evaluations were not performed in previous studies. We routinely screen for decoy cells and, if positive, measure plasma BKV‐loads. In a cohort of 203 consecutive renal transplantations performed from 2005–2008, 38 patients (19%) developed BKV‐viremia and were treated with reduction of immunosuppression. Based on subsequent allograft biopsy results and peak BKV‐viremia, patients were assigned to three groups: (i) definitive PyVAN (n = 13), (ii) presumptive PyVAN defined by plasma BKV‐loads of ≥4 log10 copies/ml (n = 17) and (iii) low BKV‐viremia (n = 8). Clearance of BKV‐viremia was achieved in 35/38 patients (92%) and subsequent clinical rejection occurred in 3/35 patients (8.6%), both without any difference among the groups. Patients with definitive PyVAN had higher peak plasma BKV‐loads and required longer time for clearance (8.8 vs. 4.6 vs. 2.9 months; p = 0.001). However, allograft function remained stable from baseline to last follow‐up at 34 months (range 18–60) in all three groups with median serum creatinine of 1.6 mg/dl, 1.6 mg/dl and 1.3 mg/dl, respectively. We conclude that screening for BKV‐replication and reduction of immunosuppression is an effective strategy to preserve medium‐term allograft function even in patients developing definitive PyVAN.

CXCR3 Mediates Renal Th1 and Th17 Immune Response in Murine Lupus Nephritis

Infiltration of T cells into the kidney is a typical feature of human and experimental lupus nephritis that contributes to renal tissue injury. The chemokine receptor CXCR3 is highly expressed on Th1 cells and is supposed to be crucial for their trafficking into inflamed tissues. In this study, we explored the functional role of CXCR3 using the MRL/MpJ-Faslpr (MRL/lpr) mouse model of systemic lupus erythematosus that closely resembles the human disease. CXCR3−/− mice were generated and backcrossed into the MRL/lpr background. Analysis of 20-wk-old CXCR3−/− MRL/lpr mice showed amelioration of nephritis with reduced glomerular tissue damage and decreased albuminuria and T cell recruitment. Most importantly, not only the numbers of renal IFN-γ-producing Th1 cells, but also of IL-17-producing Th17 cells were significantly reduced. Unlike in inflamed kidneys, there was no reduction in the numbers of IFN-γ- or IL-17-producing T cells in spleens, lymph nodes, or the small intestine of MRL/lpr CXCR3−/− mice. This observation suggests impaired trafficking of effector T cells to injured target organs, rather than the inability of CXCR3−/− mice to mount efficient Th1 and Th17 immune responses. These findings show a crucial role for CXCR3 in the development of experimental lupus nephritis by directing pathogenic effector T cells into the kidney. For the first time, we demonstrate a beneficial effect of CXCR3 deficiency through attenuation of both the Th1 and the newly defined Th17 immune response. Our data therefore identify the chemokine receptor CXCR3 as a promising therapeutic target in lupus nephritis.

Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye

Collagen VIII is localized in subendothelial and subepithelial extracellular matrices. It is a major component of Descemets membrane, a thick basement membrane under the corneal endothelium, where it forms a hexagonal lattice structure; a similar structure, albeit less extensive, may be formed in other basement membranes. We have examined the function of collagen VIII in mice by targeted inactivation of the genes encoding the two polypeptide subunits, Col8a1 and Col8a2. Analysis of these mice reveals no major structural defects in most organs, but demonstrates that type VIII collagen is required for normal anterior eye development, particularly the formation of a corneal stroma with the appropriate number of fibroblastic cell layers and Descemets membrane of appropriate thickness. Complete lack of type VIII collagen leads to dysgenesis of the anterior segment of the eye: a globoid, keratoglobus‐like protrusion of the anterior chamber with a thin corneal stroma. Descemets membrane is markedly thinned. The corneal endothelial cells are enlarged and reduced in number, and show a decreased ability to proliferate in response to different growth factors in vitro. An important function of collagen VIII may therefore be to generate a peri‐ or subcellular matrix environment that permits or stimulates cell proliferation. Hopfer, U., Fukai, N., Hopfer, H., Wolf, G., Joyce, N., Li, E., Olsen, B. R. Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye. FASEB J. 19, 1232–1244 (2005)

Chemokine Receptor CXCR3 Mediates T Cell Recruitment and Tissue Injury in Nephrotoxic Nephritis in Mice

The chemokine receptor CXCR3 is highly expressed on Th1 polarized T cells and has been predicted to play an important role in T cell recruitment and immune response in a number of inflammatory and autoimmune diseases. For testing whether CXCR3 plays a role in renal inflammation, CXCR3-deficient mice were generated and nephrotoxic nephritis was induced in C57BL/6 CXCR3(-/-) and C57BL/6 wild-type mice. Induction of the nephrotoxic nephritis leads to an increased renal mRNA expression of IP-10/CXCL10 (8.6-fold), Mig/CXCL9 (2.3-fold), and I-TAC/CXCL11 (4.9-fold) during the autologous phase at days 7 and 14. This increased chemokine expression was paralleled by the renal infiltration of T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Compared with wild-type mice, CXCR3-deficient mice had significantly reduced renal T cell infiltrates. Moreover, CXCR3(-/-) mice developed less severe nephritis, with significantly lower albuminuria, better renal function, and a reduced frequency of glomerular crescent formation. Nephritic wild-type and CXCR3(-/-) mice both elicited an efficient systemic nephritogenic immune response in terms of antigen-specific IgG production and IFN-gamma expression by splenocytes in response to the nephritogenic antigen. These findings indicate that the ameliorated nephritis in CXCR3-deficient mice is due to impaired renal trafficking of effector T cells rather than their inability to mount an efficient humoral or cellular immune response. The neutralization of CXCR3 might be a promising therapeutic strategy for Th1-dependent inflammatory renal disease.

The importance of cell-mediated immunity in the course and severity of autoimmune anti-glomerular basement membrane disease in mice

Anti‐glomerular basement membrane (GBM) disease is a rapidly progressive glomerulonephritis (GN) resulting from autoimmunity against the Goodpasture antigen α3(IV)NC1. In addition to the well‐characterized antibody contribution, a T helper 1 (Th1) response has been suspected as the culprit for glomerular injury. We induced anti‐GBM disease in DBA/1, C57BL/6, AKR, and NOD mice with recombinant human α3(IV)NC1 to investigate the involvement of humoral and cellular autoimmunity. DBA/1 mice had crescentic GN 11 wk postimmunization with α3(IV)NC1. C57BL/6 and AKR mice developed a chronic disease course resulting in comparable kidney injury to DBA/1 mice within 6 months. NOD revealed only minor glomerular changes. The rapid course and the severity of the disease in DBA/1 mice can be explained by our immunological findings in their sera and splenocytes: 1) high antibody titers specific for the putative clinically relevant epitope of α3(IV)NC1 with Th1‐type isotypes, and 2) a strong proliferative response and high amounts of the inflammatory cytokine IFN‐γ, secreted by splenocytes stimulated in vitro with α3(IV)NC1, with only low amounts of the anti‐inflammatory cytokine IL‐10. Our in vivo and in vitro results provide direct evidence that the balance between Th1 and Th2 responses associates with the outcome of anti‐GBM disease in mice.—Hopfer, H., Maron, R., Butzmann, U., Helmchen, U., Weiner, H. L., Kalluri, R. The importance of cell‐mediated immunity in the course and severity of autoimmune anti‐glomerular basement membrane disease in mice. FASEB J. 17, 860–868 (2003)

The Novel WD-repeat Protein Morg1 Acts as a Molecular Scaffold for Hypoxia-inducible Factor Prolyl Hydroxylase 3 (PHD3)

Hypoxia-inducible factor-1 (HIF-1), a transcriptional complex composed of an oxygen-sensitive α- and a β-subunit, plays a pivotal role in cellular adaptation to low oxygen availability. Under normoxia, the α-subunit of HIF-1 is hydroxylated by a family of prolyl hydroxylases (PHDs) and consequently targeted for proteasomal degradation. Three different PHDs have been identified, but the difference among their in vivo roles remain unclear. PHD3 is strikingly expressed by hypoxia, displays high substrate specificity, and has been identified in other signaling pathways. PHD3 may therefore hydroxylate divergent substrates and/or connect divergent cellular responses with HIF. We identified a novel WD-repeat protein, recently designated Morg1 (MAPK organizer 1), by screening a cDNA library with yeast two-hybrid assays. The interaction between PHD3 and Morg1 was confirmed in vitro and in vivo. We found seven WD-repeat domains by cloning the full-length cDNA of Morg1. By confocal microscopy both proteins co-localize within the cytoplasm and the nucleus and display a similar tissue expression pattern in Northern blots. Binding occurs at a conserved region predicted to the top surface of one propeller blade. Finally, HIF-mediated reporter gene activity is decreased by Morg1 and reduced to basal levels when Morg1 is co-expressed with PHD3. Suppression of Morg1 or PHD3 by stealth RNA leads to a marked increase of HIF-1 activity. These results indicate that Morg1 specifically interacts with PHD3 most likely by acting as a molecular scaffold. This interaction may provide a molecular framework between HIF regulation and other signaling pathways.

Pathology of Resolving Polyomavirus-Associated Nephropathy

Control of polyomavirus BK (BKV) is achieved by reducing immunosuppression allowing an effective BKV‐specific T‐cell response. The morphology of resolving BKV‐associated nephropathy (PyVAN) has not been systematically investigated. Ninety‐nine surveillance biopsies of 35 patients with BKV viremia treated exclusively by immunosuppression reduction were scored according to Banff criteria and grouped relative to BKV viremia as pre‐, increasing, decreasing and post‐BKV viremia. Thirty‐four of 35 patients (97%) cleared BKV viremia after a median of 9 months posttransplantation. The tubulitis score, extent of tubules with intraepithelial lymphocytes, and interstitial inflammation significantly increased from the time of increasing to decreasing viremia. Tubulointerstitial inflammation, to a lower extent, persisted after clearance. The number of SV40+ tubules correlated with the BKV load in plasma, but SV40 immunohistochemistry was frequently negative (60%). During decreasing viremia, 31% of PyVAN cases were plasma cell‐rich and 40% showed tubular HLA‐DR expression. Compared to baseline 1 month posttransplantation, allograft function remained stable or improved in 29/35 patients (83%) after a median follow‐up of 48 months. Within 1 year after clearance of BKV viremia, clinical rejection occurred in 2/35 patients (6%). Our data suggest that resolving PyVAN is typically characterized by a self‐limiting acute interstitial nephritis, morphologically indistinguishable from interstitial rejection.

Efficacy of induction therapy with ATG and intravenous immunoglobulins in patients with low-level donor-specific HLA-antibodies.

Low‐level donor‐specific HLA‐antibodies (HLA‐DSA) (i.e. detectable by single‐antigen flow beads, but negative by complement‐dependent cytotoxicity crossmatch) represent a risk factor for early allograft rejection. The short‐term efficacy of an induction regimen consisting of polyclonal anti‐T‐lymphocyte globulin (ATG) and intravenous immunoglobulins (IvIg) in patients with low‐level HLA‐DSA is unknown. In this study, we compared 67 patients with low‐level HLA‐DSA not having received ATG/IvIg induction (historic control) with 37 patients, who received ATG/IvIg induction. The two groups were equal regarding retransplants, HLA‐matches, number and class of HLA‐DSA. The overall incidence of clinical/subclinical antibody‐mediated rejection (AMR) was lower in the ATG/IvIg than in the historic control group (38% vs. 55%; p = 0.03). This was driven by a significantly lower rate of clinical AMR (11% vs. 46%; p = 0.0002). Clinical T‐cell‐mediated rejection (TCR) was significantly lower in the ATG/IvIg than in the historic control group (0% vs. 50%; p < 0.0001). Within the first year, allograft loss due to AMR occurred in 7.5% in the historic control and in 0% in the ATG/IvIg group. We conclude that in patients with low‐level HLA‐DSA, ATG/IvIg induction significantly reduces TCR and the severity of AMR, but the high rate of subclinical AMR suggests an insufficient control of the humoral immune response.

Detection of Clinical and Subclinical Tubulo-Interstitial Inflammation by the Urinary CXCL10 Chemokine in a Real-Life Setting

Urinary CXCL10 is a promising noninvasive biomarker for tubulo‐interstitial allograft inflammation, but its diagnostic characteristics have not been assessed in a real‐life setting. We investigated urinary CXCL10 in 213 consecutive renal allograft recipients having 362 surveillance biopsies at 3/6 months and 80 indication biopsies within the first year posttransplant. Allograft histology results were classified as (i) acute Banff score zero, (ii) interstitial infiltrates only, (iii) tubulitis t1, (iv) tubulitis t2–3 and (v) isolated vascular compartment inflammation. For clinical and subclinical pathologies, urinary CXCL10 correlated well with the extent of tubulo‐interstitial inflammation. To determine diagnostic characteristics of urinary CXCL10, histological groups were separated into two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (i.e. tubulitis t1–3 and isolated vascular compartment inflammation). For subclinical pathologies, AUC was 0.69 (sensitivity 61%, specificity 72%); for clinical pathologies, AUC was 0.74 (sensitivity 63%, specificity 80%). A urinary CXCL10‐guided biopsy strategy would have reduced performance of surveillance and indication biopsies by 61% and 64%, respectively. Missed (sub)clinical pathologies were mostly tubulitis t1 and isolated vascular compartment lesions. In real life, urinary CXCL10 had clinically useful diagnostic properties making it a candidate biomarker to guide allograft biopsies.