Wendy Dam

Platelets and granulocytes, in particular the neutrophils, form important compartments for circulating vascular endothelial growth factor.

The measurement of circulating vascular endothelial growth factor (VEGF) levels as a prognostic factor will gain increasing relevance in the diagnosis and evaluation of treatment in cancer patients. Angiogenesis is an absolute requirement in tumour growth and metastatic disease. In the present study data are presented which indicate that circulating VEGF mainly resides in peripheral blood cells. In 15 healthy volunteers we demonstrated that approximately 34% of the circulating VEGF resides in platelets and approximately 11% in patients with cancer (n= 4). An important part namely 58% in healthy volunteers and 69% in patients with cancer of the total circulating VEGF is contained in granulocytes, particular in the neutrophils, as confirmed by fluorescence-activated cell sorting (FACS). Also an increased VEGF level per granulocyte is found in patients with cancer (77 μg VEGF/l) compared with the healthy volunteers (164 μg VEGF/l). In contrast only 2% was present in plasma. The biological significance of platelet- or granulocyte-derived VEGF is not yet known. Liberation of VEGF from these compartments could well be of importance for tumour angiogenesis. Therefore, future studies on the clinical value of circulating VEGF as a prognostic factor in cancer patients should include measurements of VEGF in peripheral blood cells.

Mechanism of hyperthermic potentiation of cisplatin action in cisplatin-sensitive and -resistant tumour cells

In this study, the mechanism(s) by which heat increases cis-diamminedichloroplatinum (cisplatin, cDDP) sensitivity in cDDP-sensitive and -resistant cell lines of murine as well as human origin were investigated. Heating cells at 43 degrees C during cDDP exposure was found to increase drug accumulation significantly in the cDDP-resistant cell lines but had little effect on drug accumulation in the cDDP-sensitive cell lines. DNA adduct formation, however, was significantly increased in all cell lines studied. Furthermore, ongoing formation of platinum (Pt)-DNA adducts after the end of cDDP treatment was enhanced and/or adduct removal was decreased in heated cells, resulting in relatively more DNA damage remaining at 24 h after the end of cDDP exposure. Correlation plots with survival revealed weak correlations with cellular Pt accumulation (r2 = 0.59) and initial Pt-DNA adduct formation (r2 = 0.64). Strong correlations, however, were found with Pt-DNA adducts at 6 h (r2 = 0.97) and 24 h (r2 = 0.89) after the incubation with the drug. In conclusion, the mechanism by which heat sensitizes cells for cDDP action seems to be the sum of multiple factors, which comprise heat effects on accumulation, adduct formation and adduct processing. This mechanism did not seem to differ between cDDP-sensitive and -resistant cells, emphasizing the potential of hyperthermia to reduce cDDP resistance.

Role of metallothionein in cisplatin sensitivity of germ-cell tumours

Cisplatin (CDDP) is an extremely active drug in the treatment of germ‐cell tumours. Earlier, we found an unexpected inverse correlation between the total amount of sulfhydryl groups and CDDP sensitivity in a panel of 3 human germ‐cell‐tumour and 3 colon‐carcinoma cell lines. Major components of the sulfhydryl groups are glutathione and metallothionein (MT). We further investigated a possible role of MT in the CDDP sensitivity of germ‐cell tumours. MT levels and functionality of the germ‐cell‐tumour and colon‐carcinoma cell lines were found to be inversely correlated with CDDP sensitivity. No difference in sub‐cellular localization of MT could be observed among the types of cell lines. In agreement with the in vitro data, immunohistochemical detection of MT was high in 11/14 primary human germ‐cell tumours and low in 7/7 human colon carcinomas. MT‐protein expression in primary germ‐cell tumours did not discriminate between responding and non‐responding patients. As compared with the primary tumours, MT‐protein expression decreased in 5/7 post‐chemotherapy residual vital tumours or remained undetectable (2/7). MT‐protein expression in the germ‐cell tumours was not related to total p53‐protein expression. In summary, over‐expression of MT was found in germ‐cell tumours, both in cell lines and in human tumours. Although MT‐protein over‐expression seems to be associated with the CDDP sensitivity of germ‐cell tumours, MT‐protein expression in primary germ‐cell tumours did not differ between responding and non‐responding patients and therefore cannot be used to predict response to chemotherapy. Int. J. Cancer 85:777–781, 2000.

Urinary Vitamin D Binding Protein: A Potential Novel Marker of Renal Interstitial Inflammation and Fibrosis

Non-invasive tubulointerstitial damage markers may allow better titration and monitoring of renoprotective therapy. We investigated the value of urinary vitamin D binding protein excretion (uVDBP) as a tubulointerstitial inflammation and fibrosis marker in adriamycin rats, and tested whether uVDBP parallels renal damage and responds to therapy intensification in humans. In adriamycin (ADR) rats, uVDBP was strongly elevated vs controls (CON) already 6 wks after nephrosis induction (ADR: 727±674 [mean±SD] vs CON: 9±12 µg/d, p<0.01), i.e. before onset of pre-fibrotic and inflammatory tubulointerstitial damage, and at all following 6-wk time points until end of follow up at 30 wks (ADR: 1403±1026 vs CON: 206±132 µg/d, p<0.01). In multivariate regression analysis, uVDBP was associated with tubulointerstitial macrophage accumulation (standardized beta = 0.47, p = 0.01) and collagen III expression (standardized beta = 0.44, p = 0.02) independently of albuminuria. In humans, uVDBP was increased in 100 microalbuminuric subjects (44±93 µg/d) and in 47 CKD patients with overt proteinuria (9.2±13.0 mg/d) compared to 100 normoalbuminuric subjects (12±12 µg/d, p<0.001). In CKD patients, uVDBP responded to intensification of renoprotective therapy (ACEi+liberal sodium: 9.2±13.0 mg/d vs dual RAAS blockade+low sodium: 2747±4013, p<0.001), but remained still >100-fold increased during maximal therapy vs normoalbuminurics (p<0.001), consistent with persisting tubulointerstitial damage. UVDBP was associated with tubular and inflammatory damage markers KIM-1 (standardized beta = 0.52, p<0.001), beta-2-microglobuline (st.beta = 0.45, p<0.001), cystatin C (st.beta = 0.40, p<0.001), MCP-1 (st.beta = 0.31, p<0.001) and NGAL (st.beta = 0.20, p = 0.005), independently of albuminuria. UVDBP may be a novel urinary biomarker of tubulointerstitial damage. Prospectively designed studies are required to validate our findings and confirm its relevance in the clinical setting.

Improvement of in vivo transfer of plasmid DNA in muscle: comparison of electroporation versus ultrasound.

Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods—electroporation and ultrasound—that facilitate DNA uptake into cells. Mice (C57Bl/6) were injected intramuscular using plasmid DNA encoding an intracellular protein (p53) followed by electroporation or ultrasound. Then 48 hr after the injections the mice were sacrificed. The parameter for transfection efficiency was the area of muscle expressing the transgene. The p53 expression plasmid showed a 36-fold increase (p = 0.015) in transfection efficiency with electroporation compared to ultrasound. Compared with ultrasound, electroporation significantly improves transfection efficiency of naked plasmid DNA transfer into skeletal muscle.

Immunocytochemical analysis of cisplatin-induced platinum-DNA adducts with double-fluorescence video microscopy.

To detect low-level DNA platination, a sensitive immunocyto- and histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A two- to five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDP-sensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients.

Pro- and anti-apoptotic effects of p53 in cisplatin-treated human testicular cancer are cell context-dependent

In murine testicular cancer (TC) cells wild-type p53 contributes to sensitivity to DNA-damaging drugs in a dose-dependent way. In human TC, however, the role of wild-type p53 functionality in chemotherapeutic response remains elusive. We analyzed functionality of wild-type p53 in cisplatin sensitivity in the human TC setting using a p53 short interfering (si)RNA approach. The cisplatin-sensitive TC cell line (Tera), the subline with acquired cisplatin resistance (Tera-CP) and a panel of intrinsically resistant TC cell lines (Scha and 2102EP), all expressing wild-type p53, were used. p53 and p53 transcriptional targets MDM2 and p21Waf1/Cip1 (p21) were expressed in a p53 transactivation-dependent way in all TC cell lines. Following cisplatin exposure, expression levels of p53 increased, with a subsequent increase in MDM2 and p21 mRNA and protein levels and Fas cell membrane levels. Downregulation of p53 with siRNA lowered cisplatin-induced apoptosis in Tera and Tera-CP, which was associated with a diminished Fas membrane expression. In contrast, p53 suppression augmented cisplatin-induced apoptosis in Scha and 2102EP and concomitantly strongly suppressed MDM2 and p21 mRNA and protein expression. Our results indicate that p53 is involved in transactivation of pro- and anti-apoptotic genes in untreated and cisplatin-treated TC cells, but subtle differences are present between TC cell lines. The opposite role of p53 in cisplatin-induced apoptosis among TC cell lines demonstrates the importance of the cellular context for the p53 transactivation phenotype in TC cells.

Targeting tubulointerstitial remodeling in proteinuric nephropathy in rats

ABSTRACT Proteinuria is an important cause of tubulointerstitial damage. Anti-proteinuric interventions are not always successful, and residual proteinuria often leads to renal failure. This indicates the need for additional treatment modalities by targeting the harmful downstream consequences of proteinuria. We previously showed that proteinuria triggers renal lymphangiogenesis before the onset of interstitial inflammation and fibrosis. However, the interrelationship of these interstitial events in proteinuria is not yet clear. To this end, we specifically blocked lymphangiogenesis (anti-VEGFR3 antibody), monocyte/macrophage influx (clodronate liposomes) or lymphocyte and myofibroblast influx (S1P agonist FTY720) separately in a rat model to investigate the role and the possible interaction of each of these phenomena in tubulointerstitial remodeling in proteinuric nephropathy. Proteinuria was induced in 3-month old male Wistar rats by adriamycin injection. After 6 weeks, when proteinuria has developed, rats were treated for another 6 weeks by anti-VEGFR3 antibody, clodronate liposomes or FTY720 up to week 12. In proteinuric rats, lymphangiogenesis, influx of macrophages, T cells and myofibroblasts, and collagen III deposition and interstitial fibrosis significantly increased at week 12 vs week 6. Anti-VEGFR3 antibody prevented lymphangiogenesis in proteinuric rats, however, without significant effects on inflammatory and fibrotic markers or proteinuria. Clodronate liposomes inhibited macrophage influx and partly reduced myofibroblast expression; however, neither significantly prevented the development of lymphangiogenesis, nor fibrotic markers and proteinuria. FTY720 prevented myofibroblast accumulation, T-cell influx and interstitial fibrosis, and partially reduced macrophage number and proteinuria; however, it did not significantly influence lymphangiogenesis and collagen III deposition. This study showed that proteinuria-induced interstitial fibrosis cannot be halted by blocking lymphangiogenesis or the influx of macrophages. On the other hand, FTY720 treatment did prevent T-cell influx, myofibroblast accumulation and interstitial fibrosis, but not renal lymphangiogenesis and proteinuria. We conclude that tubulointerstitial fibrosis and inflammation are separate from lymphangiogenesis, at least under proteinuric conditions. Summary: Targeting lymphangiogenesis, inflammation or fibrosis separately in a rat model of proteinuric nephropathy showed that treating any of these changes alone is not effective in treating the disease.

Hepatic syndecan-1 changes associate with dyslipidemia after renal transplantation

Syndecan‐1 is a transmembrane heparan sulfate (HS) proteoglycan present on hepatocytes and involved in uptake of triglyceride‐rich lipoproteins via its HS polysaccharide side chains. We hypothesized that altered hepatic syndecan‐1 metabolism could be involved in dyslipidemia related to renal transplantation. In a rat renal transplantation model elevated plasma triglycerides were associated with fivefold increased expression of hepatic syndecan‐1 mRNA (p < 0.01), but not protein. Expression of syndecan‐1 sheddases (ADAM17, MMP9) and heparanase was significantly up‐regulated after renal transplantation (all p < 0.05). Profiling of HS side chains revealed loss of hepatic HS upon renal transplantation accompanied by significant decreased functional capacity for VLDL binding (p = 0.02). In a human renal transplantation cohort (n = 510), plasma levels of shed syndecan‐1 were measured. Multivariate analysis showed plasma syndecan‐1 to be independently associated with triglycerides (p < 0.0001) and inversely with HDL cholesterol (p < 0.0001). Last, we show a physical association of syndecan‐1 to HDL from renal transplant recipients (RTRs), but not to HDL from healthy controls. Our data suggest that after renal transplantation loss of hepatic HS together with increased syndecan‐1 shedding hampers lipoprotein binding and uptake by the liver contributing to dyslipidemia. Our data open perspectives toward improvement of lipid profiles by targeted inhibition of syndecan‐1 catabolism in renal transplantation.

Incipient renal transplant dysfunction associates with tubular syndecan-1 expression and shedding

Syndecan-1 is a transmembrane heparan sulfate proteoglycan involved in regenerative growth and cellular adhesion. We hypothesized that the induction of tubular syndecan-1 is a repair response to incipient renal damage in apparently stable, uncomplicated renal transplant recipients. We quantified tubular syndecan-1 in unselected renal protocol biopsies taken 1 yr after transplantation. Spearman rank correlation analysis revealed an inverse correlation between tubular syndecan-1 expression and creatinine clearance at the time of biopsy (r = -0.483, P < 0.03). In a larger panel of protocol and indication biopsies from renal transplant recipients, tubular syndecan-1 correlated with tubular proliferation marker Ki67 (r = 0.518, P < 0.0001). In a rat renal transplantation model, 2 mo after transplantation, mRNA expression of syndecan-1 and its major sheddase, A disintegrin and metalloproteinase-17, were upregulated (both P < 0.03). Since shed syndecan-1 might end up in the circulation, in a stable cross-sectional human renal transplant population (n = 510), we measured plasma syndecan-1. By multivariate regression analysis, we showed robust independent associations of plasma syndecan-1 with renal (plasma creatinine and plasma urea) and endothelial function parameters (plasma VEGF-A, all P < 0.01). By various approaches, we were not able to localize syndecan-1 in vessel wall or endothelial cells, which makes shedding of syndecan-1 from the endothelial glycocalyx unlikely. Our data suggest that early damage in transplanted kidneys induces repair mechanisms within the graft, namely, tubular syndecan-1 expression for tubular regeneration and VEGF production for endothelial repair. Elevated plasma syndecan-1 levels in renal transplantation patients might be interpreted as repair/survival factor related to loss of tubular and endothelial function in transplanted kidneys.